Proproteins and methods of use thereof

ABSTRACT

The present disclosure provides for proprotein and activatable proprotein compositions. A proprotein contains a functional protein (i.e. a full length protein or functional fragment thereof) which is coupled to a peptide mask that inhibits the binding of the functional protein to its target or binding partner. An activatable proprotein contains a functional protein coupled to a peptide mask, and further coupled to an activatable linker, wherein in an non-activated state, the peptide mask inhibits binding of the functional protein to its target or binding partner and in an activated state the peptide mask does not inhibit binding of the functional protein to its target or binding partner. Proproteins can provide for reduced toxicity and adverse side effects that could otherwise result from binding of a functional protein at non-treatment sites if it were not inhibited from binding its binding partner. Proproteins can further provide improved biodistribution characteristics. Proproteins containing a peptide mask can display a longer in vivo or serum half-life than the corresponding functional protein not containing a peptide mask. The disclosure further provides methods of screening for, making, and using these proproteins.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/154,730, filed Feb. 23, 2009, which application is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

Protein-based therapies have changed the face of medicine, findingapplication in a variety of different diseases. As with any therapies,however, the need and desire for improved specificity and selectivityfor targets is of great interest.

In the realm of small molecule drugs, strategies have been developed toprovide prodrugs of an active chemical entity. Such prodrugs areadministered in a relatively inactive (or significantly less active)form. Once administered, the prodrug is metabolized in vivo into theactive compound. Such prodrug strategies can provide for increasedselectivity of the drug for its intended target and for a reduction ofadverse effects. Drugs used to target hypoxic cancer cells, through theuse of redox-activation, utilize the large quantities of reductaseenzyme present in the hypoxic cell to convert the drug into itscytotoxic form, essentially activating it. Since the prodrug has lowcytotoxicity prior to this activation, there is a markedly decreasedrisk of damage to non-cancerous cells, thereby providing for reducedside-effects associated with the drug. There is a need in the field fora strategy for providing features of a prodrug to protein-basedtherapeutics, especially in developing second generation of proteindrugs having known targets to which they bind. Increased targeting tothe disease site could reduce systemic mechanism-based toxicities andlead to broader therapeutic utility.

SUMMARY OF THE INVENTION

The present disclosure provides for proprotein and activatableproprotein compositions.

In one aspect the present disclosure provides for a compositioncomprising a functional protein that is not an antibody or an antibodyfragment, wherein the functional protein is coupled to a peptide maskthat: (i) inhibits binding of the functional protein to its bindingpartner and (ii) does not have an amino acid sequence of the bindingpartner. In one embodiment, the functional protein is further coupled toa cleavable linker capable of being cleaved, such that: (i) in anuncleaved state, the peptide mask inhibits binding of the functionalprotein to its binding partner and (ii) in a cleaved state, the peptidemask does not inhibit binding of the functional protein to its bindingpartner. In one embodiment, the cleavable linker is capable of beingspecifically cleaved by an enzyme, capable of being reduced by areducing agent, or capable of being photolysed. In one embodiment, thecleavable linker is capable of being specifically cleaved by an enzymeat a rate of at least 5×10⁴ M⁻¹S.

In another embodiment, the peptide mask is recombinantly expressed. Inone embodiment, the peptide mask is unique for the functional protein.

In another embodiment, the peptide mask has a therapeutic effect onceuncoupled from the functional protein.

In one embodiment, the peptide mask is 8-15 amino acids in length.

In one embodiment, the peptide mask has less than 50% amino acidsequence homology to its binding partner.

In one embodiment, the peptide mask contains less than 50% geneticallynon-encoded amino acids. In a related embodiment, the geneticallynon-encoded amino acids are D-amino acids, β-amino acids, or γ-aminoacids.

In one embodiment the functional protein is a full-length protein, afunctional fragment of a full-length protein, a globular protein, afibrous protein, or a multimeric protein. In a specific embodiment, thefunctional protein is a ligand. In a related embodiment, the ligand isan interferon protein and is selected from the group consisting ofinterferon type I, interferon type II, and interferon type III or isselected from the group consisting of IFN-α, IFN-β and IFN-ω. In aspecific embodiment, the interferon protein is IFN-α. In a specificembodiment, the IFN-α protein is selected from the group consisting of2a, 2b, and con1. In a related embodiment, the binding partner is areceptor for the interferon protein. In such an embodiment, the receptorfor the interferon protein is selected from the group consisting ofIFNAR, IFNAR1, IFNAR2, IFNGR, and IFNLR1. In a related embodiment, thepeptide mask contains a sequence selected from those presented in Table3 or a sequence at least having 90% homology thereof. In a specificembodiment, the peptide mask contains the consensus sequenceTDVDYYREWXXXXXXXX.

In another embodiment, the functional protein is a soluble membraneprotein or a functional fragment thereof. In another embodiment, thefunctional protein is a soluble receptor or fragment thereof. In arelated embodiment, the functional protein is the extracellular domainof a receptor protein or a fraction thereof. In specific embodiments,the peptide mask inhibits the binding of the soluble receptor to itsligand or the peptide mask inhibits the receptor's ligand bindingdomain. In a more specific embodiment, the receptor is Notch and can beselected from the group consisting Notch1, Notch2, Notch3 and Notch4. Ina related embodiment, the Notch ligand is selected from the groupconsisting DLL1, DLL3, DLL4, Jagged1, and Jagged2. In a specificembodiment, the peptide mask contains a sequence selected from thosepresented in Table 14 or a sequence having at least 90% homologythereof.

In other embodiments, the cleavable linker is a substrate for an enzymeselected from the substrates in Table 2. In related embodiments, thecleavable linker is a substrate for an enzyme selected from the groupconsisting of matriptase, plasmin, MMP-9, uPA, HCV-NS3/4, PSA, andlegumain, or specifically is a substrate for matriptase or HCV-NS3/4. Inone embodiment, the consensus sequence for a matriptase substratecomprises XXQAR(A/V)X or AGPR. In another embodiment, the consensussequence for a HCV-NS3/4 substrate comprises DEXXXC(A/S) or DLXXXT(A/S).In another embodiment, the sequence for an MMP-9 substrate comprisesVHMPLGFLGP. In another embodiment, the sequence for a plasmin substratecomprises QGPMFKSLWD.

In another embodiment the composition further contains an Fc region ofan immunoglobulin.

In yet another embodiment, the coupling of the peptide mask to thefunctional protein is non-covalent.

In some embodiments, the peptide mask inhibits binding of the functionalprotein to its binding partner allosterically. In other embodiments, thepeptide mask inhibits binding of the functional protein to its bindingpartner sterically.

In most embodiments, the binding affinity of the peptide mask to thefunctional protein is less than the binding affinity of the bindingpartner to the functional protein. In a specific embodiment, thedissociation constant (K_(d)) of the peptide mask towards the functionalprotein is at least 100 times greater than the K_(d) of the functionalprotein towards its binding partner. In a more specific embodiment, theK_(d) of the peptide mask towards the functional protein is lower thanabout 5 nM.

In another embodiment, when the composition is not in the presence of anenzyme capable of cleaving the cleavable linker, the peptide maskinhibits the binding of the functional protein to its binding partner byat least 90% when compared to when the composition is in the presence ofthe enzyme capable of cleaving the cleavable linker and the peptide maskdoes not inhibit the binding of the functional protein to its bindingpartner.

In another aspect, the present disclosure provides for a pharmaceuticalcomposition, wherein said pharmaceutical composition comprises atherapeutically effective amount of a composition comprising afunctional protein that is not an antibody or an antibody fragment,wherein the functional protein is coupled to a peptide mask that: (i)inhibits binding of the functional protein to its binding partner and(ii) does not have an amino acid sequence of the binding partner and apharmaceutically acceptable excipient. In one specific embodiment ofthis pharmaceutical composition, the functional protein is furthercoupled to a cleavable linker capable of being cleaved, such that: (i)in an uncleaved state, the peptide mask inhibits binding of thefunctional protein to its binding partner and (ii) in a cleaved state,the peptide mask does not inhibit binding of the functional protein toits binding partner.

In another aspect, the present disclosure also provides a method oftreating a disease or disorder, wherein a pharmaceutical compositioncomprising a therapeutically effective amount of a compositioncomprising a functional protein that is not an antibody or an antibodyfragment, wherein the functional protein is coupled to a peptide maskthat: (i) inhibits binding of the functional protein to its bindingpartner and (ii) does not have an amino acid sequence of the bindingpartner and a pharmaceutically acceptable excipient is administered. Inone specific embodiment of this method, the functional protein isfurther coupled to a cleavable linker capable of being cleaved, suchthat: (i) in an uncleaved state, the peptide mask inhibits binding ofthe functional protein to its binding partner and (ii) in a cleavedstate, the peptide mask does not inhibit binding of the functionalprotein to its binding partner. In a specific embodiment, the disease ordisorder is cancer. In another specific embodiment, the disease ordisorder is a liver condition such as Hepatitis C infection orhepatocellular cancer. In yet another specific embodiment, the diseaseor disorder involves angiogenesis.

In another aspect, the present disclosure provides for a librarycomprising a plurality of candidate activatable functional proteins,displayed on the surface of a replicable biological entity. In oneembodiment, the functional protein is an interferon or a soluble Notchreceptor protein.

In another aspect, the present disclosure provides a method of making alibrary of candidate peptide masks, comprising: introducing into genomesof replicable biological entities a collection of recombinant DNAconstructs that each encode a peptide mask, said introducing producingrecombinant replicable biological entities; and culturing saidrecombinant replicable biological entities under conditions suitable forexpression and display of the candidate peptide masks. In a relatedembodiment, the candidate peptide masks are screened for the ability tobind an interferon protein or a soluble Notch receptor. In a specificembodiment, the interferon protein is pro-IFN-α.

In another aspect, the present disclosure provides a method of screeningfor a peptide mask, said method comprising: contacting a plurality ofcandidate peptide masks with a functional protein; and screening a firstpopulation of members with a functional protein; wherein said methodprovides for selection of peptide masks. In one embodiment, thecandidate peptide masks are screened for the ability to bind aninterferon protein or a soluble Notch receptor. In a specificembodiment, interferon protein is pro-IFN-α.

In another aspect, the present disclosure provides a method of screeningfor an activatable functional protein coupled to a peptide mask, saidmethod comprising: contacting a plurality of candidate activatableproteins with a binding partner capable of binding the functionalprotein and an enzyme capable of cleaving a cleavable linker of theactivatable protein; screening a first population of members of saidplurality which bind to said binding partner in the presence of theenzyme; contacting said first population with the binding partner in theabsence of the enzyme; and screening a second population of members fromsaid first population by depleting said first population for membersthat bind the binding partner in the absence of the enzyme, wherein saidmethod provides for selection of candidate activatable functionalproteins which exhibit decreased binding to its binding partner in theabsence of the enzyme as compared to binding partner binding in thepresence of the enzyme. In one embodiment, the candidate peptide masksare screened for the ability to bind an interferon protein or a solubleNotch receptor. In one specific embodiment, the interferon protein ispro-IFN-α.

In another aspect, the present disclosure provides a method of making alibrary of candidate activatable functional proteins, each coupled to apeptide mask, said method comprising: introducing into genomes ofreplicable biological entities a collection of recombinant DNAconstructs that encode a plurality of candidate activatable functionalproteins, said introducing producing recombinant replicable biologicalentities; and culturing said recombinant replicable biological entitiesunder conditions suitable for expression and display of the candidateactivatable functional proteins. In one embodiment, the candidateactivatable functional proteins differ in the sequence of their coupledpeptide masks. In a specific embodiment, the functional protein is aninterferon or a soluble Notch receptor protein.

In another aspect, the present disclosure provides a method of screeningfor an activatable functional protein coupled to a peptide, said methodcomprising: contacting a plurality of candidate activatable proteinswith a binding partner capable of binding the functional protein and anenzyme capable of cleaving a cleavable linker of the activatableprotein; screening a first population of members of said plurality whichbind to said binding partner in the presence of the enzyme; contactingsaid first population with the binding partner in the absence of theenzyme; and screening a second population of members from said firstpopulation by depleting said first population for members that bind thebinding partner in the absence of the enzyme; wherein said methodprovides for selection of candidate activatable functional proteinswhich exhibit decreased binding to its binding partner in the absence ofthe enzyme as compared to binding partner binding in the presence of theenzyme. In one embodiment, the functional protein is an interferon or asoluble Notch receptor protein.

In another aspect, the present disclosure provides a vector encoding afunctional protein and a peptide mask wherein the peptide mask iscapable of inhibiting the functional protein's ability to bind itsbinding partner.

In one embodiment, the functional protein is an interferon protein or asoluble Notch receptor protein.

In one specific aspect the present disclosure provides a modified IFN-αprotein comprising a substrate capable of cleavage by matriptase.

In another specific aspect the present disclosure provides a modifiedIFN-α protein comprising a substrate capable of cleavage by HCV-NS3/4.

In another specific aspect the present disclosure provides a modifiedsoluble Notch receptor protein comprising a substrate capable ofcleavage by a matrix metalloproteinase.

In another specific aspect the present disclosure provides a modifiedsoluble Notch receptor protein comprising a substrate capable ofcleavage by plasmin.

In another specific aspect the present disclosure provides a modifiedsoluble Notch receptor protein comprising a substrate capable ofcleavage by legumain.

In another specific aspect the present disclosure provides a modifiedsoluble Notch receptor protein comprising a substrate capable ofcleavage by uPA.

In another specific aspect the present disclosure provides a modifiedsoluble Notch receptor protein comprising a substrate capable ofcleavage by PSA.

In another aspect the present disclosure provides a protein therapeuticfor the treatment of Hepatitis C having an improved bioavailabilitycomprising a functional protein coupled to a peptide mask and acleavable linker, wherein the affinity of binding of the proteintherapeutic to its target is higher in liver tissue when compared to thebinding of the protein therapeutic to its target in a non-liver tissue,wherein target is present in both tissues. In one embodiment, thecleavable linker comprises a substrate specific for a matriptase or HCVNS3/4 enzyme.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts an exemplary masked activatable folded proprotein. Thefigures display a protein not capable of binding partner due toInteraction with specific and unique peptide mask.

FIG. 2 depicts enrichment of IFN-α binding peptides for use as masks, asassayed by FACS.

FIG. 3 depicts the binding of two pro-IFN-α molecules, pro-IFN-α-47 andpro-IFN-α-49CS, before and after treatment with MMP-9.

FIG. 4 depicts testing of individual clones for binding to human Notch 1EGF-like domains 11-13.

DETAILED DESCRIPTION OF THE INVENTION Proproteins Introduction andGeneral Features

The present disclosure provides for proproteins.

The proprotein compositions described herein contain a full lengthprotein or a functional fragment of a full-length protein (collectivelyreferred to as ‘functional protein’ herein) coupled to a peptide mask.The peptide mask can inhibit binding of the functional protein to itsbinding partner or target (binding partner and target usedinterchangeably herein). The peptide mask can inhibit binding of thefunctional protein to its binding partner sterically or allosterically.Generally, the functional protein displays two distinct levels ofbinding to its binding partner, based on the presence and/or location ofthe peptide mask.

When a functional protein is coupled to a peptide mask and is in thepresence of its binding partner, specific binding of the functionalprotein to its binding partner can be reduced or inhibited, as comparedto the specific binding of the functional protein to its binding partnernot coupled to the peptide mask.

A functional protein is a full-length protein or functional fragmentthereof and has functional activity or physiological activity (e.g., invivo or in vitro), such as, for example, binding affinity to a target orbinding partner, capability of effecting signaling pathways, hasenzymatic activity, or the like. A functional protein fragment alsoretains functional activity or physiological activity (e.g., in vivo orin vitro). Such activity can be, for example, retaining relevantbiological activity of the full length protein, i.e. binding, targeting,signaling, triggering a particular signaling cascade, modulating aparticular pathway, and the like.

In one embodiment the functional protein is not an antibody or anantibody fragment.

A functional protein of the present invention can be naturally occurringor non-naturally occurring.

The proproteins of the present invention or the functional protein canbe post-translationally modified.

A functional protein can be globular, fibrous, or multimeric.

A functional protein can be an ligand, an extracellular ligand, such as,for example a interferon protein, or more specifically, for example, anIFN-α full length protein, an IFN-β full length protein, an IFN-γ fulllength protein, or a IFN-ψ0 full length protein.

A functional protein can be a soluble membrane protein, for example, asoluble receptor, for example a soluble Notch Receptor, for exampleNotch1, Notch2, Notch3, or Notch4 receptor.

A functional protein can be taken up intracellularly or can remainextracellular.

Proproteins of the present invention can contain naturally occurringamino acids or non-naturally occurring amino acids, or both. Proproteinsof the present invention can contain L-amino acids, D-amino acids, or amixture of both. In specific embodiments, the functional proteins of thepresent invention can be coupled to peptide masks that contain naturallyoccurring or non-naturally occurring amino acids, or both.

Proproteins of the present invention can contain a mutated variant of anaturally occurring full length protein or functional protein fragment.That is, a functional protein can be a mutant of a naturally occurringprotein.

The proproteins of the present invention can be synthetically generated.

The proproteins of the present invention can be recombinantly expressed,and purified.

The present disclosure further also provides activatable proproteins.

An activatable proprotein comprises a functional protein or functionalfragment thereof, coupled to a peptide mask, and further coupled to anactivatable moiety (or activatable linker such as a cleavable linker),wherein in an uncleaved state the peptide mask inhibits binding of theprotein to its binding partner and in a cleaved state the peptide maskdoes not inhibit binding of the protein to a binding partner.

The activatable moiety or activatable linker of activatable proproteincompositions, when activated, can change the conformation of the peptidemask in relationship to the functional protein. By activating theactivatable linker, the functional protein can have a different bindingaffinity to its binding partner or target.

In some instances, the activatable linker is a cleavable linker,containing a substrate capable of being specifically cleaved by anenzyme, protease, or peptidase. In other instances the activatablelinker is reducible by a reducing agent. In yet other instances, theactivatable linker is a photo-sensitive substrate, capable of beingactivated by photolysis. As used herein cleavage is used interchangeablyto denote activation by an enzyme, a reducing agent, or photolysis.

A schematic of an activatable proprotein is provided in FIG. 1. Asillustrated, the elements of the activatable proprotein are arranged sothat in an uncleaved state (or relatively inactive state) binding of theprotein to the target binding partner is inhibited due to the masking ofthe protein by the peptide mask.

By activatable it is meant that the proprotein exhibits a first level ofbinding to a binding partner when in a native or non-activated state(i.e., a first conformation), and a second level of binding to a bindingpartner in the activated state (i.e., a second conformation), whereinthe second level of binding is greater than the first level of binding.In general, access of a binding partner to the functional protein isgreater in the presence of an enzyme/reducing agent/light capable ofactivating the activatable linker than in the absence of suchenzyme/reducing agent/light. Thus, in the non-activated or uncleavedstate the protein is masked from target binding (i.e., the firstconformation is such that the peptide mask inhibits access of thebinding partner to the protein), and in the activated state the proteinis unmasked to the binding partner.

When the functional protein is coupled to both a peptide mask and anactivatable moiety, and is in the presence of its binding partner butnot in the presence of sufficient enzyme/reductase/light to activate theactivatable moiety, specific binding of the functional protein to itsbinding partner is inhibited, as compared to the specific binding of thefunctional protein to its binding partner when in the presence ofsufficient enzyme/reductase/light to activate the activatable moiety.

Proproteins can provide for reduced toxicity and/or adverse side effectsthat could otherwise result from binding of a functional protein atnon-treatment sites if it were not inhibited from binding its bindingpartner. Proproteins can provide for improved biodistributioncharacteristics. Proproteins containing a masked protein can display alonger in vivo or serum half-life than the corresponding unmaskedprotein.

In general, a proprotein can be designed by selecting a full length orfunctional fragment of a protein of interest, and constructing theremainder of the proprotein so that, when conformationally constrained,the peptide mask sterically or allosterically provides for masking ofthe binding site of the protein. Structural design criteria can be takeninto account to provide for the masking feature. Preferably, theproprotein is genetically encoded and recombinantly expressed, but canalso be synthetically produced

Proproteins exhibiting an activatable phenotype of a desired dynamicrange for target binding in a cleaved versus uncleaved conformation areprovided. Dynamic range generally refers to a ratio of (a) a detectedlevel of a parameter under a first set of conditions to (b) a detectedvalue of that parameter under a second set of conditions. For example,in the context of a proprotein, the dynamic range refers to the ratio of(a) a detected level of target protein binding to a proprotein in thepresence of an enzyme such as a protease capable of cleaving thecleavable linker of the proprotein to (b) a detected level of targetprotein binding to a proprotein in the absence of the protease. Thedynamic range of a proprotein can be calculated as the ratio of theequilibrium dissociation constant of a proprotein cleaving agent (e.g.,enzyme) treatment to the equilibrium dissociation constant of theproprotein cleaving agent treatment. The greater the dynamic range of aproprotein, the better the activatable phenotype of the proprotein.Proproteins having relatively higher dynamic range values (e.g., greaterthan 1, 2, 3, 4, 5, or more) exhibit more desirable activatingphenotypes such that target protein binding by the proprotein occurs toa greater extent (e.g., predominantly occurs) in the presence of acleaving agent (e.g., enzyme) capable of cleaving the cleavable linkerof the proprotein than in the absence of a cleaving agent.

Activatable proproteins can be provided in a variety of structuralconfigurations. Exemplary formulae for proproteins are provided below.It is specifically contemplated that the N- to C-terminal order of thefunctional protein, the peptide mask, and the cleavable linker may bereversed within a proprotein. It is also specifically contemplated thatthe cleavable linker and peptide mask may overlap in amino acidsequence, e.g., such that the cleavable linker is contained within thepeptide mask.

For example, proproteins can be represented by the following formula (Inorder from an amino (N) terminal region to carboxyl (C) terminal region.

-   -   (peptide mask)-(linker)-(functional protein)    -   (functional protein)-(linker)-(peptide mask)    -   (peptide mask)-(activatable linker)-(functional protein)    -   (functional protein)-(activatable linker)-(peptide mask)

It should be noted that although the peptide mask and cleavable linkerare indicated as distinct components in the formula above, in allexemplary embodiments disclosed herein it is contemplated that the aminoacid sequences of the peptide mask and the cleavable linker couldoverlap, e.g., such that the cleavable linker is completely or partiallycontained within the peptide mask. In addition, the formulae aboveprovide for additional amino acid sequences that may be positionedN-terminal or C-terminal to the proprotein elements.

In many embodiments it may be desirable to insert one or more linkers,e.g., flexible linkers, into the proprotein construct so as to providefor flexibility at one or more of the peptide mask-activatable/cleavablelinker junction, the activatable/cleavable linker-protein junction, orboth. For example, the functional protein, peptide mask, and/oractivatable/cleavable linker may not contain a sufficient number ofamino acid residues (e.g., Gly, Ser, Asp, Asn, especially Gly and Ser,particularly Gly) to provide the desired flexibility. The linkers maycomprise stretches of amino acids that are or that are not naturallyoccurring. As such, the activatable phenotype of such proproteinconstructs may benefit from introduction of one or more amino acids toprovide for a flexible linker.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), (GSGGS)_(n)and (GGGS)_(n), where n is an integer of at least one), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers known inthe art. Glycine and glycine-serine polymers are relativelyunstructured, and therefore may be able to serve as a neutral tetherbetween components. Glycine accesses significantly more phi-psi spacethan even alanine, and is much less restricted than residues with longerside chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).Exemplary flexible linkers include, but are not limited toGly-Gly-Ser-Gly, Gly-Gly-Ser-Gly-Gly, Gly-Ser-Gly-Ser-Gly,Gly-Ser-Gly-Gly-Gly, Gly-Gly-Gly-Ser-Gly, Gly-Ser-Ser-Ser-Gly, and thelike. The ordinarily skilled artisan will recognize that design of aproprotein can include linkers that are all or partially flexible, suchthat the linker can include a flexible linker as well as one or moreportions that confer less flexible structure to provide for a desiredproprotein structure.

Linkers suitable for use in proproteins are generally ones that provideflexibility of the proprotein to facilitate a masked conformation. Suchlinkers are generally referred to as flexible linkers. Suitable linkerscan be readily selected and can be of different lengths, such as from 1amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 aminoacids, from 3 amino acids to 12 amino acids, including 4 amino acids to10 amino acids, amino acids to 9 amino acids, 6 amino acids to 8 aminoacids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

For example, proproteins containing these optional flexible linkers canbe represented by the following formulas (in order from an amino (N)terminal region to carboxyl (C) terminal region.

(peptide mask)-(optional flexible linker)-(activatable linker)-(optionalflexible linker)-(functional protein)

(functional protein)-(optional flexible linker)-(activatablelinker)-(optional flexible linker)-(peptide mask)

In addition to the elements described above, the proproteins can becoupled to additional elements or extra features, such as an additionaltherapeutic moiety, a targeting moiety to facilitate delivery to a cellor tissue of interest, a moiety to direct binding to a target receptorto facilitate localization of the proprotein, a Fc region of animmunoglobulin to increase serum half-life of the proprotein, forexample, and the like.

For example, proproteins containing these optional additional elementsor features can be represented by the following formulas (in order froman amino (N) terminal region to carboxyl (C) terminal region).

(targeting moiety for cellular uptake)-(peptide mask)-(activatablelinker)-(functional protein)

-   -   (functional protein)-(activatable linker)-(peptide        mask)-(targeting moiety for cellular uptake)        -   (Fc)-(peptide mask)-(activatable linker)-(functional            protein)        -   (functional protein)-(activatable linker)-(peptide            mask)-(Fc)

The dissociation constant (K_(d)) of the functional protein towards itsbinding partner when coupled to a peptide mask is greater than the K_(d)of the functional protein towards its binding partner when not coupledto a peptide mask. Conversely, the binding affinity of the functionalprotein towards its binding partner when coupled to a peptide mask islower than the binding affinity of the functional protein towards itsbinding partner when not coupled to a peptide mask.

The K_(d) of the peptide mask towards the functional protein isgenerally greater than the K_(d) of the functional protein towards itsbinding partner. Conversely, the binding affinity of the peptide masktowards the functional protein is generally lower than the bindingaffinity of the functional protein towards its binding partner.

The peptide mask can inhibit the binding of the functional protein toits binding partner. The peptide mask can bind a binding domain of thefunctional protein and inhibit binding of the functional protein to itsbinding partner. The peptide mask can sterically interfere with thebinding of the functional protein to its binding partner. The peptidemask can allosterically inhibit the binding of the functional protein toits binding partner. In these embodiments when the functional protein ismodified or coupled to a peptide mask and in the presence of bindingpartner, there is no binding or substantially no binding of thefunctional protein to its binding partner as compared to the binding ofthe functional protein not coupled to a peptide mask. This can bemeasured in vivo or in vitro in a Mask Efficiency Assay, animmunoabsorbant assay, as described herein.

When a functional protein is coupled to a peptide mask, the peptide maskcan ‘mask’ or reduce, or inhibit the specific binding of the functionalprotein to its binding partner. When a functional protein is coupled toa peptide mask, such coupling or modification can effect a structuralchange which reduces or inhibits the ability of the functional proteinto specifically bind its binding partner.

The disclosure further provides methods of use, methods of screening,and methods of making peptide-masked functional proteins.

The components of the proprotein compositions provided herein aredescribed in greater detail following.

Functional Proteins and Binding Partners

The present disclosure provides for a full-length protein or afunctional protein fragment coupled to a peptide mask that inhibits thefunctional protein from interacting with a binding partner or target.The functional proteins for use contemplated by the present disclosurecan be any full length protein or functional fragment thereof (referredto interchangeably as ‘functional proteins’). By functional protein, itis indicated that the full length protein, or functional fragmentthereof, retains relevant biological activity, i.e. binding, targeting,signaling, etc. Once unmasked, the binding of the functional protein toits binding partner or target can provide for a desired biologicaleffect, e.g., inhibition of activity of the target protein and/ordetection of a target protein. Once unmasked, a functional protein canbind to one binding partner or multiple binding partners.

The functional protein can be a naturally or non-naturally occurringprotein.

The functional protein can be recombinantly expressed, geneticallyencoded, and/or post translationally modified. The functional proteincan be synthetically constructed.

The functional protein can be a mutant of a naturally occurring protein.The mutated functional protein can retain no more than 95%, 90%, 80%,75%, 70,%, 60%, 50%, 40%, 30%, 25%, or 20% nucleic acid or amino acidsequence homology to the non-mutated functional protein.

The functional protein can be globular, fibrous, or multimeric. Thefunctional protein can exhibit folding, and can exhibit primary,secondary, or quaternary structure.

The functional protein can be a ligand, for example, an interferonprotein, for example an IFN-α protein (type 2a, 2b or con1),IFN-βprotein, IFN-γ protein, or an IFN-ω protein. The functional proteincan be a soluble membrane protein, for example, a soluble receptor, forexample a soluble Notch Receptor (for example Notch1, Notch2, Notch3, orNotch4 receptor).

The functional protein can be designed to remain extracellularly ordesigned for cellular uptake in its unmasked state.

Throughout the present disclosure the terms binding partner and targetare used interchangeably. The binding partner of the functional proteincan be extracellular, intracellular, or a transmembrane protein. In oneembodiment its binding partner of the functional protein is anextracellular protein, such as a ligand or a soluble receptor. Inanother embodiment the binding partner of the functional protein is anintracellular protein and the functional protein is capable of cellularuptake and is designed to be unmasked inside a cell. In anotherembodiment, the binding partner of the functional protein is amembrane-associated receptor.

Exemplary binding partners/targets are interferon protein receptors, orspecifically IFNAR, IFNAR1, IFNAR2, and IFNLR1. Other exemplary bindingpartner/targets are Notch ligands such as DLL1, DLL3, DLL4, Jagged1, andJagged 2.

A functional protein of the invention can specifically bind to itstarget or binding partner with a dissociation constant (K_(d)) of nomore than 1000 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 μM, 400 μM, 350μM, 300 μM, 250 μM, 200 μM, 150 μM, 100 μM, 50 μM, 25 μM, 10 μM, 5 μM, 1μM, 0.5 μM, or 0.1 μM.

In certain embodiments the functional protein coupled with a peptidemask is not an antibody or antibody fragment.

Exemplary sources for the functional protein to generateinterferon-related proproteins contemplated are provided in Table 1.

TABLE 1 Exemplary Sources for Interferon-related proproteinsPeginterferon Lambda PEGASYS (Peginterferon alfa-2a) Peginterferon(Rebetol) Actimmune (Interferon γ 1b) Avonex (Interferon β1a) Betaseron(Interferon β 1b) Rebif (Interferon β 1a) INTRON A (Interferon α-2b)PegIntron (Peginterferon α-2b)

Peptide Masks

The present disclosure provides for a functional protein coupled to apeptide mask (also interchangeably referred to as a masking peptide or amasking moiety) which inhibits the functional protein from interactingwith a binding partner. The peptide mask can specifically interact withthe functional protein and reduce or inhibit the interaction between thefunctional protein and its binding partner.

When the functional protein is in a ‘masked’ state, even in the presenceof a binding partner for the functional protein, the peptide maskinterferes with or inhibits the binding of the functional protein to itsbinding partner. However, in the unmasked state of the functionalprotein, the peptide mask's interference with target binding to thefunctional protein is reduced, thereby allowing greater access of thefunctional protein to the target and providing for target binding.

For example, when the proprotein comprises an activatable moiety, thefunctional protein can be unmasked upon cleavage of the activatablemoiety, in the presence of enzyme, preferably a disease-specific enzyme.Thus, the peptide mask is one that when the proprotein is uncleavedprovides for masking of the functional protein from target binding, butdoes not substantially or significantly interfere or compete for bindingof the target to the functional protein when the proprotein is in thecleaved conformation. Thus, the combination of the peptide mask and theactivatable moiety facilitates the switchable/activatable phenotype,with the peptide mask decreasing binding of target when the proproteinis uncleaved, and cleavage of the activatable moiety by proteaseproviding for increased binding of target.

The structural properties of the peptide mask can vary according to avariety of factors such as the minimum amino acid sequence required forinterference with protein binding to target, the target protein-proteinbinding pair of interest, the size of the functional protein, the lengthof the activatable moiety, whether the activatable moiety is positionedwithin the peptide mask and also serves to mask the functional proteinin the uncleaved proprotein, the presence or absence of linkers, thepresence or absence of a cysteine within or flanking the functionalprotein that is suitable for providing an activatable moiety of acysteine-cysteine disulfide bond, and the like.

In one embodiment, the peptide mask can be coupled to the functionalprotein by covalent binding. In another embodiment, the functionalprotein is prevented from binding to its target by binding the peptidemask to an N-terminus of the functional protein. In yet anotherembodiment, the functional protein is coupled to the peptide mask bycysteine-cysteine disulfide bridges between the peptide mask and thefunctional protein.

The peptide mask can be provided in a variety of different forms. Thepeptide mask can be selected from a known binding partner of thefunctional protein, provided that the peptide mask binds the functionalprotein with less affinity and/or avidity than the target protein towhich the functional protein is designed to bind, following cleavage ofthe activatable moiety so as to reduce interference of peptide mask intarget-protein binding. Stated differently, as discussed above, thepeptide mask is one that masks the functional protein from targetbinding when the proprotein is uncleaved, but does not substantially orsignificantly interfere or compete for binding for target when theproprotein is in the cleaved conformation.

Generally, the peptide mask is unique for the functional protein ofinterest. Examples of peptide masks that specifically interact with thefunctional protein of the proprotein include peptide masks that werespecifically screened to bind a binding domain of the functional proteinor protein fragment. Methods for screening peptide masks to obtainpeptide masks unique for the functional protein and those thatspecifically and/or selectively bind a binding domain of a bindingpartner/target are provided herein and can include protein displaymethods.

The present disclosure provides for peptide masks that can specificallyinhibit the interaction between the functional protein and its bindingpartner. Each peptide mask has a certain binding affinity for thefunctional protein. The binding affinity is generally lower than thebinding affinity between the functional protein and its binding partner.

The peptide mask of the present disclosure generally refers to an aminoacid sequence coupled to a functional protein and is positioned suchthat it reduces the functional protein's ability to specifically bindits binding partner. In some cases the peptide mask is coupled to thefunctional protein by way of a linker.

When the functional protein is coupled to a peptide mask and is in thepresence of its binding partner, specific binding of the functionalprotein to its binding partner can be reduced or inhibited, as comparedto the specific binding of the functional protein not coupled to apeptide mask or the specific binding of the parental protein to itsbinding partner. When the functional protein is coupled to both anactivatable moiety and a peptide mask and is in the presence of itsbinding partner but not sufficient enzyme or enzyme activity to cleavethe activatable moiety, specific binding of the modified protein to itsbinding partner is reduced or inhibited, as compared to the specificbinding of the functional protein coupled to an activatable moiety and apeptide mask in the presence of its binding partner and sufficientenzyme/enzyme activity/reducing agent/reducing agent activity/light toactivate the activatable moiety.

The peptide mask can inhibit the binding of the functional protein toits binding partner. The peptide mask can bind the binding domain of thefunctional protein and inhibit binding of the functional protein to itsbinding partner. The peptide mask can sterically inhibit the binding ofthe functional protein to its binding partner. The peptide mask canallosterically inhibit the binding of the functional protein to itsbinding partner.

When a functional protein is coupled to a peptide mask and in thepresence of binding partner, there is no binding or substantially nobinding of the functional protein to the binding partner, or no morethan 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, or 50% binding of the functional protein to itsbinding partner, as compared to the binding of the functional proteinnot coupled to a peptide mask, the binding of the parental protein, orthe binding of the functional protein not coupled to a peptide mask toits binding partner, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48,60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days,or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or greater when measuredin vivo or in a Mask Efficiency Assay, an in vitro immunoabsorbantassay, as described herein.

The peptide mask can be a synthetically produced string of amino acidsthat are capable of inhibiting the interaction of a functional proteinwith its binding partner. The peptide mask can be part of a linker oractivatable moiety. In related embodiments the peptide mask can beselected in an unbiased manner upon screening for specific and selectivebinding to the functional protein.

In certain embodiments, the peptide mask can have at least partial orcomplete amino acid sequence of a naturally occurring binding partner ofthe functional protein. The peptide mask can be a fragment of anaturally occurring binding partner. The fragment can retain no morethan 95%, 90%, 80%, 75%, 70,%, 60%, 50%, 40%, 30%, 25%, or 20% nucleicacid or amino acid sequence homology to the naturally occurring bindingpartner.

In some instances the peptide mask has an amino acid sequence that isnot naturally occurring or does not contain the amino acid sequence of anaturally occurring binding partner or target protein. In certainembodiments the peptide mask is not a natural binding partner of thefunctional protein. The peptide mask may be a modified binding partnerfor the functional protein which contains amino acid changes that atleast slightly decrease affinity and/or avidity of binding to thefunctional protein. In some embodiments the peptide mask contains no orsubstantially no nucleic acid or amino acid homology to the functionalprotein's natural binding partner. In other embodiments the peptide maskis no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, or 80% similar to the natural binding partner of thefunctional protein.

The present disclosure also provides for variants for a given peptidemask. The sequence of the peptide masks can be varied to retain at least95%, 90%, 80%, 75%, 70,%, 60%, 50%, 40%, 30%, 25%, or 20% nucleic acidor amino acid sequence homology to the peptide mask. Such sequencevariations may afford an improved masking ability.

The efficiency of the peptide mask to inhibit the binding of thefunctional protein to its target when coupled can be measured by aMasking Efficiency Assay, using an in vitro immunoabsorbant assay, asdescribed herein in the Examples section of the disclosure. Maskingefficiency of peptide masks is determined by at least two parameters:affinity of the peptide mask for the functional protein and the spatialrelationship of the peptide mask relative to the binding interface ofthe functional protein to its target.

Regarding affinity, by way of example, a peptide mask may have highaffinity but only partially inhibit the binding site on the functionalprotein, while another peptide mask may have a lower affinity for thefunctional protein but fully inhibit target binding. For short timeperiods, the lower affinity peptide mask may show sufficient masking; incontrast, over time, that same peptide mask may be displaced by thetarget (due to insufficient affinity for the functional protein).

In a similar fashion, two peptide masks with the same affinity may showdifferent extents of masking based on how well they promote inhibitionof the binding site on the functional protein or prevention of thefunctional protein from binding its target. In another example, apeptide mask with high affinity may bind and change the structure of thefunctional protein so that binding to its target is completely inhibitedwhile another peptide mask with high affinity may only partially inhibitbinding. As a consequence, discovery of an effective peptide mask isoften not based only on affinity but can include an empirical measure ofMasking Efficiency. The time-dependent target displacement of thepeptide mask in the functional protein can be measured to optimize andselect for peptide masks. A novel Masking Efficiency Assay is describedherein for this purpose.

A peptide mask can be identified and further optimized through ascreening procedure from a library of candidate proproteins havingvariable peptide masks. For example, a functional protein andactivatable moiety can be selected to provide for a desiredenzyme/target combination, and the amino acid sequence of the peptidemask can be identified by the screening procedure described below toidentify a peptide mask that provides for a switchable phenotype. Forexample, a random peptide library (e.g., from about 2 to about 40 aminoacids or more) may be used in the screening methods disclosed herein toidentify a suitable peptide mask. In specific embodiments, peptide maskswith specific binding affinity for a functional protein can beidentified through a screening procedure that includes providing alibrary of peptide scaffolds consisting of candidate peptide maskswherein each scaffold is made up of a transmembrane protein and thecandidate peptide mask. The library is then contacted with an entire orportion of a protein such as a full length protein, a naturallyoccurring protein fragment, or a non-naturally occurring fragmentcontaining a protein (also capable of binding the binding partner ofinterest), and identifying one or more candidate peptide masks havingdetectably bound protein. Screening can include one more rounds ofmagnetic-activated sorting (MACS) or fluorescence-activated sorting(FACS). Screening can also included determination of the dissociationconstant (K_(d)) of peptide mask towards the functional protein andsubsequent determination of the Masking Efficiency.

In this manner, proproteins having a peptide mask that inhibits bindingof the functional protein to its binding partner in an non-activatedstate and allows binding of the functional protein to its bindingpartner in a activated state can be identified, and can further providefor selection of a proprotein having an optimal dynamic range for theswitchable phenotype. Methods for identifying proproteins having adesirable switching phenotype are described in more detail herein.Alternatively, the peptide mask may not specifically bind the functionalprotein, but rather interfere with protein-binding partner bindingthrough non-specific interactions such as steric hindrance. For example,the peptide mask may be positioned in the uncleaved proprotein such thatthe tertiary or quaternary structure of the proprotein allows thepeptide mask to mask the functional protein through charge-basedinteraction, thereby holding the peptide mask in place to interfere withbinding partner access to the functional protein.

Proproteins can also be provided in a conformationally constrainedstructure, such as a cyclic structure, to facilitate the switchablephenotype. This can be accomplished by including a pair of cysteines inthe proprotein construct so that formation of a disulfide bond betweenthe cysteine pairs places the proprotein in a loop or cyclic structure.Thus the proprotein remains cleavable by the desired protease whileproviding for inhibition of target binding to the functional protein.Upon activation of the activatable moiety, the cyclic structure isopened, allowing access of binding partner to the functional protein.

The cysteine pairs can be positioned in the proprotein at any positionthat provides for a conformationally constrained proprotein, but that,following activatable moiety reduction, does not substantially orsignificantly interfere with target binding to the functional protein.For example, the cysteine residues of the cysteine pair are positionedin the peptide mask and a linker flanked by the peptide mask andprotein, within a linker flanked by the peptide mask and protein, orother suitable configurations. For example, the peptide mask or a linkerflanking a peptide mask can include one or more cysteine residues, whichcysteine residue forms a disulfide bridge with a cysteine residuepositioned opposite the peptide mask when the proprotein is in a foldedstate. It is generally desirable that the cysteine residues of thecysteine pair be positioned outside the functional protein so as toavoid interference with target binding following cleavage of theproprotein. Where a cysteine of the cysteine pair to be disulfide bondedis positioned within the functional protein, it is desirable that it bepositioned to as to avoid interference with protein-target bindingfollowing exposure to a reducing agent.

In certain embodiments, once an activatable proprotein is activated, thepeptide mask is uncoupled from the functional protein, whereby unmaskingthe functional protein. In some embodiments, once uncoupled from thefunctional protein and in a free state, the peptide has biologicalactivity or a therapeutic effect, such as binding capability. Forexample, the free peptide can bind with the same or a different bindingpartner. In certain embodiments the free peptide mask (uncoupled peptidemask) can exert a therapeutic effect, providing a secondary function tothe compositions of this invention.

The peptide masks contemplated by this disclosure can range from 1-50amino acids; in some instances can be at least than 3, 4, 5, 6, 7, 8, 9,10, 12, 15, 20, 30, or 40 amino acids, or no greater than 40, 30, 20,15, 12, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids. In specific embodimentsthe peptide masks of the present invention are 8-15 amino acids inlength.

The peptide masks of the present invention can contain geneticallyencoded or genetically non-encoded amino acids. Examples of geneticallynon-encoded amino acids are but not limited to D-amino acids, β-aminoacids, and γ-amino acids. In specific embodiments, the peptide maskscontain no more than 50%, 40%, 30%, 20%, 15%, 10%, 5% or 1% ofgenetically non-encoded amino acids.

The dissociation constant (K_(d)) of the functional protein towards thetarget or binding partner when coupled to a peptide mask can be at least5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000,100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 orgreater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000,10-1,000,000, 10-10,000,000,100-1,000,100-10,000,100-100,000,100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000,1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000,10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 timesgreater than the K_(d) of the functional protein towards its bindingpartner when not coupled to a peptide mask or the parental protein.Conversely, the binding affinity of the functional protein towards itsbinding partner when coupled to a peptide mask can be at least 5, 10,25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000,500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, orbetween 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000,10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000,100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000,1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000,100,000-1,000,000, or 100,000-10,000,000 times lower than the bindingaffinity of the functional protein towards its binding partner when notcoupled to a peptide mask.

The K_(d) of the peptide mask towards the functional protein isgenerally greater than the K_(d) of the functional protein towards itsbinding partner. The K_(d) of the peptide mask towards the functionalprotein can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500,5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times greater thanthe K_(d) of the functional protein towards its binding partner.Conversely, the binding affinity of the peptide mask towards thefunctional protein is generally lower than the binding affinity of thefunctional protein towards its binding partner. The binding affinity ofpeptide mask towards the functional protein can be at least 5, 10, 25,50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 oreven 10,000,000 times lower than the binding affinity of the functionalprotein towards its binding partner.

When the functional protein is coupled to a peptide mask and is in thepresence of the binding partner, specific binding of the functionalprotein to its binding partner can be reduced or inhibited, as comparedto the specific binding of the functional protein not coupled to apeptide mask to its binding partner. When compared to the binding of thefunctional protein not coupled to a peptide mask to its binding partner,the functional protein's ability to bind the binding partner whencoupled to a peptide mask can be reduced by at least 50%, 60%, 70%, 80%,90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96, hours, or 5, 10, 15,30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12 months or greater when measured in vivo or in a Mask EfficiencyAssay, an in vitro immunoabsorbant assay, as described herein.

The peptide mask can inhibit the binding of the functional protein toits binding partner. The peptide mask can bind a binding domain of thefunctional protein and inhibit binding of the functional protein to itsbinding partner. The peptide mask can sterically interfere with thebinding of the functional protein to its binding partner. The peptidemask can allosterically inhibit the binding of the functional protein toits binding partner. In these embodiments when the functional protein iscoupled to a peptide mask and in the presence of binding partner, thereis no binding or substantially no binding of the functional protein toits binding partner, or no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50%binding of the functional protein to its binding partner, as compared tothe binding of the functional protein not coupled to a peptide mask, orthe functional protein not coupled to a peptide mask to its bindingpartner, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84,96, hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 months or greater when measured in vivo orin a Masking Efficiency Assay, as described herein.

When a functional protein is coupled to or coupled to a peptide mask,the peptide mask can ‘mask’ or reduce, or inhibit the specific bindingof the functional protein to its binding partner. When a functionalprotein is coupled to or coupled to a peptide mask, such coupling ormodification can effect a structural change which reduces or inhibitsthe ability of the functional protein to specifically bind its bindingpartner.

A functional protein coupled to or coupled to a peptide mask can berepresented by the following formulae (in order from an amino (N)terminal region to carboxyl (C) terminal region. As depicted in theformula, it may be further desirable to insert one or more linkers, e.g.flexible linkers, in to the composition to provide for increasedflexibility.

-   -   (peptide mask)-(functional protein)    -   (functional protein)-(peptide mask)    -   (peptide mask)-(linker)-(functional protein)    -   (functional protein)-(linker)-(peptide mask)

Exemplary peptide masks can contain sequences as presented in Tables 3and 14. A peptide mask of the invention can contain a sequence selectedfrom those presented in Table 3 or a sequence at least having 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or 95% homology thereof. A peptide mask ofthe invention can contain a sequence selected from those presented inTable 14 or a sequence at least having 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or 95% homology thereof.

An exemplary peptide mask can contain the consensus sequenceTDVDYYREWXXXXXXXX.

Other exemplary peptide masks can be specific for an interferon protein,for example an IFN-α protein (type 2a, 2b or con1), IFN-β protein, IFN-γprotein, or an IFN-ω protein. Other exemplary peptide masks can bespecific for a Notch Receptor, for example Notch1, Notch2, Notch3, orNotch4 receptor.

Activatable Moieties

The present invention provides for activatable proproteins containingboth a peptide mask and an activatable moiety or domain which modulatesthe proprotein's ability to bind its binding partner. Such compositionsare referred to as activatable proproteins.

By activatable it is meant that the proprotein exhibits a first level ofbinding to a binding partner when in a native (e.g., uncleaved state)(i.e., a first conformation), and a second level of binding to itsbinding partner in the activated (e.g., cleaved state) (i.e., a secondconformation). The second level of binding partner binding is greaterthan the first level of binding.

For example, a proprotein can comprise a full-length protein orfunctional fragment thereof, a peptide mask and an activatable moietythat modulates the functional protein's ability to bind its target orbinding partner. The activatable moiety can be a cleavable linker. Insuch an example, in an uncleaved state, the functional protein iscoupled to the peptide mask and the peptide mask interferes with thefunctional protein's ability to bind its binding partner but in acleaved state, the functional protein is uncoupled and the functionalprotein can interact with its binding partner. Methods for screening forsubstrates for enzymes that can be utilized as cleavable linkersaccording to the present invention are described herein.

The cleavable linkers of the present disclosure may include an aminoacid sequence that can serve as a substrate for a protease, reductase,or photolysis. The cleavable linker is positioned in the maskedfunctional protein such that when the linker is cleaved by a such as anenzyme or a protease in the presence of a binding partner, resulting ina cleaved state, the functional protein binds the binding partner, andin an uncleaved state, in the presence of the binding partner, bindingof the functional protein to its binding partner is inhibited by thepeptide mask. It should be noted that the amino acid sequence of thecleavable linker may overlap with or be included within the peptidemask, such that all or a portion of the cleavable linker facilitates“masking” of the functional protein when the proprotein is in theuncleaved conformation.

In general, access of binding partner to the functional protein isgreater in the presence of an enzyme capable of cleaving the cleavablelinker than in the absence of such an enzyme. Thus, in the native oruncleaved state the proprotein is prevented from binding to its partner(i.e., the first conformation is such that it interferes with access ofthe binding partner to the proprotein), and in the cleaved state thefunctional protein is unmasked to binding its partner.

The activatable moiety may be selected based on a protease that isco-localized in tissue with the desired binding partner of thefunctional protein. A variety of different conditions are known in whicha binding partner of interest is co-localized with a protease, where thesubstrate of the protease is known in the art. In the example of cancer,the binding partner tissue can be a cancerous tissue, particularlycancerous tissue of a solid tumor. There are reports in the literatureof increased levels of proteases having known substrates in a number ofcancers, e.g., solid tumors. See, e.g., La Rocca et al, (2004) BritishJ. of Cancer 90(7): 1414-1421. Non-liming examples of disease include:all types of cancers (breast, lung, colorectal, prostate, head and neck,pancreatic, etc), rheumatoid arthritis, Crohn's disease, melanomas, SLE,cardiovascular damage, ischemia, etc. Furthermore, anti-angiogenictargets, such as VEGF, are known. As such, where the functional proteinis selected such that it is capable of binding an anti-angiogenic targetsuch as Notch 1, a suitable activatable moiety will be one whichcomprises a peptide substrate that is cleavable by a protease that ispresent at the cancerous treatment site, particularly that is present atelevated levels at the cancerous treatment site as compared tonon-cancerous tissues. In one exemplary embodiment, a functional proteincan bind an Interferon receptor and the activatable moiety can be amatrix metalloprotease (MMP) substrate, and thus is cleavable by an MMP.In other embodiments, the functional protein can bind a target ofinterest and the activatable moiety can be, for example, legumain,plasmin, matriptase, HCV-NS3/4, TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin,caspase, human neutrophil elastase, beta-secretase, uPA, or PSA. Inother embodiments, the proprotein is activated by other disease-specificproteases, in diseases other than cancer such as Hepatitis C.

The unmodified or uncleaved activatable moiety can allow for efficientinhibition or masking of the functional protein by tethering the peptidemask to the functional protein. When the activatable moiety is modified(cleaved, reduced, photolysed), the functional protein is no longerinhibited or unmasked and can bind its binding partner.

The activatable moiety is capable of being specifically modified(cleaved, reduced or photolysed) by an agent (i.e. enzyme, reducingagent, light) at a rate of about 0.001−1500×10⁴ M⁻¹S⁻¹ or at least0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50,75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500×10⁴ M⁻¹S⁻¹.

For specific cleavage by an enzyme, contact between the enzyme andactivatable moiety is made. When the proprotein comprising a functionalprotein coupled to a peptide mask and an activatable moiety is in thepresence of target and sufficient enzyme activity, the activatablemoiety can be cleaved. Sufficient enzyme activity can refer to theability of the enzyme to make contact with the activatable moiety andeffect cleavage. It can readily be envisioned that an enzyme may be inthe vicinity of the activatable moiety but unable to cleave because ofother cellular factors or protein modification of the enzyme.

Exemplary substrates can include but are not limited to substratescleavable by one or more of the following enzymes or proteases in Table2.

TABLE 2 Exemplary Enzymes/Proteases ADAM10 Caspase 8 Cathepsin S MMP 8ADAM12 Caspase 9 FAP MMP 9 ADAM17 Caspase 10 Granzyme B MMP-13 ADAMTSCaspase 11 Guanidinobenzoatase (GB) MMP 14 ADAMTS5 Caspase 12 HepsinMT-SP1 BACE Caspase 13 Human Neutrophil Neprilysin Elastase (HNE)Caspases Caspase 14 Legumain HCV-NS3/4 Caspase 1 Cathepsins Matriptase 2Plasmin Caspase 2 Cathepsin A Meprin PSA Caspase 3 Cathepsin B MMP 1PSMA Caspase 4 Cathepsin D MMP 2 TACE Caspase 5 Cathepsin E MMP 3 TMPRSS3/4 Caspase 6 Cathepsin K MMP 7 uPA Caspase 7 MT1-MMP neurosin calpaintPA HCV-NS3/4A

Exemplary consensus sequences for specific enzymes are presented inTables 11 and 12. In one embodiment the consensus sequence for amatriptase substrate comprises XXQAR(A/V)X or AGPR. In anotherembodiment the consensus sequence for a HCV-NS3/4 substrate comprisesDEXXXC(A/S) or DLXXXT(A/S).

In one embodiment the sequence for a MMP-9 substrate is VHMPLGFLGP. Inanother embodiment the sequence for a plasmin substrate is QGPMFKSLWD.

Identifying and Optimizing Proproteins and Components Thereof

Methods for identifying and/or optimizing proproteins and componentsthereof, as well as compositions useful in such methods, are describedbelow.

Libraries of Candidate Proproteins and their Components, and Display onReplicable Biological Entities

In general, the screening methods to identify a proprotein, itscomponents such as the peptide mask/peptide and the cleavable linkerand/or to optimize a proprotein for an activatable phenotype involveproduction of a library of replicable biological entities (asexemplified by cells) that display on their surface a plurality ofdifferent candidate proproteins. These libraries can then be subjectedto screening methods to identify candidate proproteins and componentshaving one or more desired characteristics of a proprotein and itscomponents.

The candidate proprotein libraries can contain candidate proproteinsthat differ by one or more of the peptide mask, linker (which may bepart of the peptide mask), cleavable linker (which may be part of thepeptide mask), and protein. To identify candidate peptide masks orpeptides, the candidate proproteins in the library are variable for thepeptide mask and/or the linker.

Suitable replicable biological entities include cells (e.g., bacteria(e.g., E. coli), yeast (e.g., S. cerevisiae), mammalian cells),bacteriophage, and viruses. Bacterial host cells and bacteriophage,particularly bacterial host cells, are of interest.

A variety of display technologies using replicable biological entitiesare known in the art. These methods and entities include, but are notlimited to, display methodologies such as mRNA and ribosome display,eukaryotic virus display, and phage, bacterial, yeast, and mammaliancell surface display. See Wilson, D. S., et al. 2001 PNAS USA98(7):3750-3755; Muller, O. J., et al. (2003) Nat. Biotechnol. 3:312;Bupp, K. and M. J. Roth (2002) Mol. Ther. 5(3):329 3513; Georgiou, G.,et al., (1997) Nat. Biotechnol. 15(1):29 3414; and Boder, E. T. and K.D. Wittrup (1997) Nature Biotech. 15(6):553 557. Surface display methodsare attractive since they enable application of fluorescence-activatedcell sorting (FACS) for library analysis and screening. See Daugherty,P. S., et al. (2000) J. Immuunol. Methods 243(1 2):211 2716; Georgiou,G. (2000) Adv. Protein Chem. 55:293 315; Daugherty, P. S., et al. (2000)PNAS USA 97(5):2029 3418; Olsen, M. J., et al. (2003) Methods Mol. Biol.230:329 342; Boder, E. T. et al. (2000) PNAS USA 97(20):10701 10705;Mattheakis, L. C., et al. (1994) PNAS USA 91(19): 9022 9026; and Shusta,E. V., et al. (1999) Curr. Opin. Biotech. 10(2):117 122. Exemplary phagedisplay and cell display compositions and methods are described in U.S.Pat. Nos. 5,223,409; 5,403,484; 7,118,879; 6,979,538; 7,208,293;5,571,698; and 5,837,500. Additional display methodologies which may beused to identify a peptide capable of binding to a biological target ofinterest are described in U.S. Pat. No. 7,256,038, the disclosure ofwhich is incorporated herein by reference.

Optionally, the display scaffold can include a protease cleavage site(different from the protease cleavage site of the cleavable linker) toallow for cleavage of a proprotein or candidate proprotein from asurface of a host cell.

Methods of making a proprotein libraries and/or candidate proproteinlibraries comprises: (a) constructing a set of recombinant DNA vectorsas described below that encode a plurality of proproteins and/orcandidate proproteins; (b) transforming host cells with the vectors ofstep (a); and (c) culturing the host cells transformed in step (b) underconditions suitable for expression and display of the fusionpolypeptides.

Constructs Encoding Candidate Proproteins and Candidate ProproteinComponents

The disclosure further provides vectors and nucleic acid constructswhich include sequences coding for proproteins and/or candidateproproteins. Suitable nucleic acid constructs include, but are notlimited to, constructs which are capable of expression in prokaryotic oreukaryotic cells. Expression constructs are generally selected so as tobe compatible with the host cell in which they are to be used. Incertain embodiments, the vector encodes a protein and a peptide mask ora protein, a peptide mask, and a cleavable linker.

For example, non-viral and/or viral constructs vectors may be preparedand used, including plasmids, which provide for replication ofproprotein- or candidate proprotein-encoding DNA and/or expression in ahost cell. The choice of vector will depend on the type of cell in whichpropagation is desired and the purpose of propagation. Certainconstructs are useful for amplifying and making large amounts of thedesired DNA sequence. Other vectors are suitable for expression in cellsin culture. The choice of appropriate vector is well within the skill ofthe art. Many such vectors are available commercially. Methods forgenerating constructs can be accomplished using methods well known inthe art.

In order to effect expression in a host cell, the polynucleotideencoding a proprotein or candidate proprotein is operably linked to aregulatory sequence as appropriate to facilitate the desired expressionproperties. These regulatory sequences can include promoters, enhancers,terminators, operators, repressors, and inducers. Expression constructsgenerally also provide a transcriptional and translational initiationregion as may be needed or desired, which may be inducible orconstitutive, where the coding region is operably linked under thetranscriptional control of the transcriptional initiation region, and atranscriptional and translational termination region. These controlregions may be native to the species from which the nucleic acid isobtained, or may be derived from exogenous sources.

Constructs, including expression constructs, can also include aselectable marker operative in the host to facilitate, for example,growth of host cells containing the construct of interest. Suchselectable marker genes can provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture.

Production of Nucleic Acid Sequences Encoding Candidate Proproteins

Production of candidate proproteins for use in the screening methods canbe accomplished using methods known in the art. Polypeptide display,single chain antibody display, antibody display and antibody fragmentdisplay are methods well know in the art. In general, an element of aproprotein e.g., peptide mask, to be varied in the candidate proproteinlibrary is selected for randomization. The candidate proproteins in thelibrary can be fully randomized, partially randomized or biased in theirrandomization, e.g. in nucleotide/residue frequency generally or inposition of amino acid(s) within an element. For example, the proproteinelement (e.g., candidate peptide mask) can be partially randomized so asto provide for only a subset of amino acids at a selected position(e.g., to provide for a flexible linker at a selected position in theamino acid sequence, to provide for an amino acid residue of a desiredcharacteristic (e.g., hydrophobic, polar, positively charged, negativelycharged, etc.). In another example, the proprotein element (e.g.,candidate peptide mask) can be partially randomized so that one or moreresidues within the otherwise randomized amino acid sequence is selectedand held as invariable among a population or subpopulation of proproteinlibrary members (e.g., so as to provide a cysteine at a desired positionwithin the candidate peptide mask).

Methods of Screening for Proproteins and Components Thereof Methods ofScreening for Peptide Masks

Generally, the method for screening for peptide masks and peptide maskshaving a desired masking phenotype is accomplished through a positivescreening step (to identify members that bind the functional protein)and a negative screening step (to identify members that do not bind thefunctional protein). The negative screening step can be accomplished by,for example, depleting from the population members that bind thefunctional protein in the absence of the peptide mask. It should benoted that the library screening methods described herein can beinitiated by conducting the negative screening first to select forcandidates that do not bind the functional protein and then conductingthe positive screening (i.e., exposing library of replicable biologicalentities displaying candidate peptide masks to a functional protein andselecting for members which bind the functional protein.).

The positive and negative screening steps can be conveniently conductedusing flow cytometry to sort candidate masks based on binding of adetectably labeled functional protein. One “round” or “cycle” of thescreening procedure involves both a positive selection step and anegative selection step. The methods may be repeated for a library suchthat multiple cycles (including complete and partial cycles, e.g., 1.5cycles, 2.5 cycles, etc.) are performed. In this manner, members of theplurality of candidate masks that exhibit binding to the functionalprotein of interest may be enriched in the resulting population.

Proprotein Mask Efficiency Assay: Choosing an effective peptide mask isnot necessarily based solely on affinity but can include an empiricalmeasure of ‘masking efficiency.’ Two exemplary assays can be used. Thefirst is the measurement of the affinity of a Proprotein binding to acell surface displaying a candidate peptide mask by, for example, FACS.In the second assay the ability of a peptide mask to inhibit Proproteinbinding to its binding partner at therapeutically relevantconcentrations and times can be measured. For this second method, animmunoabsorbant assay (MEA, Mask Efficiency Assay) to measure thetime-dependent binding of proprotein binding to its binding partner hasbeen developed.

Choosing an effective peptide mask cannot be based solely on affinitybut must include an empirical measure of masking efficiency. To do thiswe have used two assays. The first is the measurement of the affinity ofprotein binding to the cell surface displayed peptide mask by FACS. Inthe second assay we measure the ability of a peptide mask to inhibitproprotein binding to its target at therapeutically relevantconcentrations and times. To do this we developed an immunoabsorbantassay (MEA, Masking efficiency assay) to measure the time dependentbinding partner displacement of the peptide mask in the Proproteincontext.

In general, the screening methods are conducted by first generating anucleic acid library encoding a plurality of candidate masks in adisplay scaffold, which is in turn introduced into a display scaffoldfor expression on the surface of a replicable biological entity.

Prior to the screening method, it may be desirable to enrich for cellsexpressing an appropriate peptide display scaffold on the cell surface.The optional enrichment allows for removal of cells from the celllibrary that (1) do not express peptide display scaffolds on the cellouter membrane or (2) express non-functional peptide display scaffoldson the cell outer membrane. By “non-functional” is meant that thepeptide display scaffold does not properly display a candidate mask,e.g., as a result of a stop codon or a deletion mutation.

Enrichment for cells can be accomplished by growing the cell populationand inducing expression of the peptide display scaffolds. The cells arethen sorted based on, for example, detection of a detectable signal ormoiety incorporated into the scaffold or by use of a detectably-labeledantibody that binds to a shared portion of the display scaffold or theproprotein. These methods are described in greater detail in U.S. Pat.No. 7,256,038 and U.S. Patent Application Publication No: 2007/0065878,published Mar. 22, 2007 and are incorporated by reference in theirentirety.

Methods of Screening for Protease Substrates for Use as CleavableLinkers

In general, the method for screening for candidate substrates to achievethe desired activatable phenotype for the proprotein is accomplishedthrough a positive screening step (to identify members cleave thesubstrate following exposure to enzyme) and a negative screening step(to identify members that do not cleave the substrate when exposed toenzyme). The negative screening step can be accomplished by, forexample, depleting from the population members that cleave the substrateabsence of the protease. It should be noted that the library screeningmethods described herein can be initiated by conducting the negativescreening first to select for candidates that do not cleave thesubstrate in the absence of enzyme treatment, and then conducting thepositive screening (i.e., treating with enzyme and selecting for memberswhich cleave the substrate.

The positive and negative screening steps can be conveniently conductedusing flow cytometry to sort candidate substrates based on cleavage. One“round” or “cycle” of the screening procedure involves both a positiveselection step and a negative selection step. The methods may berepeated for a library such that multiple cycles (including complete andpartial cycles, e.g., 1.5 cycles, 2.5 cycles, etc.) are performed. Inthis manner, members of the plurality of candidate substrates thatexhibit the activating characteristics may be enriched in the resultingpopulation.

In general, the screening methods are conducted by first generating anucleic acid library encoding a plurality of candidate substrates in adisplay scaffold, which is in turn introduced into a display scaffoldfor expression on the surface of a replicable biological entity.

Prior to the screening method, it may be desirable to enrich for cellsexpressing an appropriate peptide display scaffold on the cell surface.The optional enrichment allows for removal of cells from the celllibrary that (1) do not express peptide display scaffolds on the cellouter membrane or (2) express non-functional peptide display scaffoldson the cell outer membrane. By “non-functional” is meant that thepeptide display scaffold does not properly display a candidatesubstrate, e.g., as a result of a stop codon or a deletion mutation.

Enrichment for cells can be accomplished by growing the cell populationand inducing expression of the peptide display scaffolds. The cells arethen sorted based on, for example, detection of a detectable signal ormoiety incorporated into the scaffold or by use of a detectably-labeledantibody that binds to a shared portion of the display scaffold or theproprotein. These methods are described in greater detail in U.S. Pat.No. 7,256,038 and U.S. Patent Application Publication No: 2007/0065878,published Mar. 22, 2007 and are incorporated by reference in theirentirety.

Methods of Screening for Activatable Proproteins

In general, the method for screening for candidate proproteins having adesired activatable phenotype is accomplished through a positivescreening step (to identify members that bind a binding partnerfollowing exposure to enzyme) and a negative screening step (to identifymembers that do not bind a binding partner when not exposed to enzyme).The negative screening step can be accomplished by, for example,depleting from the population members that bind the binding partner inthe absence of the protease. It should be noted that the libraryscreening methods described herein can be initiated by conducting thenegative screening first to select for candidates that do not bindlabeled binding partner in the absence of enzyme treatment (i.e., do notbind labeled binding partner when not cleaved), and then conducting thepositive screening (i.e., treating with enzyme and selecting for memberswhich bind labeled binding partner in the cleaved state).

The positive and negative screening steps can be conveniently conductedusing flow cytometry to sort candidate proproteins based on binding of adetectably labeled binding partner. One “round” or “cycle” of thescreening procedure involves both a positive selection step and anegative selection step. The methods may be repeated for a library suchthat multiple cycles (including complete and partial cycles, e.g., 1.5cycles, 2.5 cycles, etc.) are performed. In this manner, members of theplurality of candidate proproteins that exhibit the activatingcharacteristics of a proprotein may be enriched in the resultingpopulation.

In general, the screening methods are conducted by first generating anucleic acid library encoding a plurality of candidate proproteins in adisplay scaffold, which is in turn introduced into a display scaffoldfor expression on the surface of a replicable biological entity.

Prior to the screening method, it may be desirable to enrich for cellsexpressing an appropriate peptide display scaffold on the cell surface.The optional enrichment allows for removal of cells from the celllibrary that (1) do not express peptide display scaffolds on the cellouter membrane or (2) express non-functional peptide display scaffoldson the cell outer membrane. By “non-functional” is meant that thepeptide display scaffold does not properly display a candidateproprotein, e.g., as a result of a stop codon or a deletion mutation.

Enrichment for cells can be accomplished by growing the cell populationand inducing expression of the peptide display scaffolds. The cells arethen sorted based on, for example, detection of a detectable signal ormoiety incorporated into the scaffold or by use of a detectably-labeledantibody that binds to a shared portion of the display scaffold or theproprotein. These methods are described in greater detail in U.S. Pat.No. 7,256,038 and U.S. Patent Application Publication No: 2007/0065878,published Mar. 22, 2007 and are incorporated by reference in theirentirety.

Detectable Labels

As used herein, the terms “label”, “detectable label” and “detectablemoiety” are used interchangeably to refer to a molecule capable ofdetection, including, but not limited to, radioactive isotopes,fluorescers, chemiluminescers, chromophores, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions,metal sols, ligands (e.g., biotin, avidin, streptavidin or haptens) andthe like. The term “fluorescer” refers to a substance or a portionthereof which is capable of exhibiting fluorescence in the detectablerange. Exemplary detectable moieties suitable for use as labels include,affinity tags and fluorescent proteins.

Any fluorescent polypeptide (also referred to herein as a fluorescentlabel) well known in the art is suitable for use as a detectable moietyor with an affinity tag of the peptide display scaffolds describedherein. A suitable fluorescent polypeptide will be one that can beexpressed in a desired host cell, such as a bacterial cell or amammalian cell, and will readily provide a detectable signal that can beassessed qualitatively (positive/negative) and quantitatively(comparative degree of fluorescence). Exemplary fluorescent polypeptidesinclude, but are not limited to, yellow fluorescent protein (YFP), cyanfluorescent protein (CFP), GFP, mRFP, RFP (tdimer2), HCRED, etc., or anymutant (e.g., fluorescent proteins modified to provide for enhancedfluorescence or a shifted emission spectrum), analog, or derivativethereof. Further suitable fluorescent polypeptides, as well as specificexamples of those listed herein, are provided in the art and are wellknown.

Biotin-based labels also find use in the methods disclosed herein.Biotinylation of target molecules and substrates is well known, forexample, a large number of biotinylation agents are known, includingamine-reactive and thiol-reactive agents, for the biotinylation ofproteins, nucleic acids, carbohydrates, carboxylic acids; see, e.g.,chapter 4, Molecular Probes Catalog, Haugland, 6th Ed. 1996, herebyincorporated by reference. A biotinylated substrate can be detected bybinding of a detectably labeled biotin binding partner, such as avidinor streptavidin. Similarly, a large number of haptenylation reagents arealso known.

Screening Methods

Any suitable method that provides for separation and recovery ofproproteins of interest may be utilized. For example, a cell displayinga proprotein of interest may be separated by FACS, immunochromatographyor, where the detectable label is magnetic, by magnetic separation. As aresult of the separation, the population is enriched for cells thatexhibit the desired characteristic, e.g., exhibit binding to bindingpartner following cleavage or have decreased or no detectable binding tobinding partner in the absence of cleavage.

For example, selection of candidate proproteins having bound detectablylabeled binding partner can be accomplished using a variety oftechniques known in the art. For example, flow cytometry (e.g., FACS®)methods can be used to sort detectably labeled candidate proproteinsfrom unlabeled candidate proproteins. Flow cytometry methods can beimplemented to provide for more or less stringent requirements inseparation of the population of candidate proproteins, e.g., bymodification of gating to allow for “dimmer” or to require “brighter”cell populations in order to be separated into the second population forfurther screening.

In another example, immunoaffinity chromatography can be used toseparate target-bound candidate proproteins from those that do not bindtarget. For example, a support (e.g., column, magnetic beads) havingbound anti-target antibody can be contacted with the candidateproproteins that have been exposed to protease and to binding partner.Candidate proproteins having bound target bind to the anti-targetantibody, thus facilitating separation from candidate proproteinslacking bound target. Where the screening step is to provide for apopulation enriched for uncleaved candidate proproteins that haverelatively decreased target binding or no detectable target binding(e.g., relative to other candidate proproteins), the subpopulation ofinterest is those members that lack or have a relatively decreaseddetectably signal for bound target. For example, where an immunoaffinitytechnique is used in such negative selection for bound target, thesubpopulation of interest is that which is not bound by the anti-targetsupport.

Therapeutic Uses of Proproteins

Proproteins described herein can be selected for use in methods oftreatment of suitable subjects according to the cleavable linker-proteincombination provided. Exemplary non-limiting uses for proproteins arefor hepatitis C, cancer, and angiogenesis. For example, a patientsuffering from a condition (e.g., such as described above) can beadministered a therapeutically effective amount of a proprotein.

Use of a proprotein can allow for decreased dosing frequency compared tothe unmodified or parent protein.

The proprotein can be administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local injection (e.g., at the site of asolid tumor). Parenteral administration routes include intramuscular,intravenous, intraarterial, intraperitoneal, or subcutaneousadministration.

The appropriate dosage of proprotein will depend on the type of diseaseto be treated, the severity and course of the disease, the patient'sclinical history and response to the proprotein, and the discretion ofthe physician. Proproteins can suitably be administered to the patientat one time or over a series of treatments.

Depending on the type and severity of the disease, about 1 ug/kg to 100mg/kg, or at least 1 ug/kg, 5 ug/kg, 10 ug/kg, 50 ug/kg, 100 ug/kg, 250ug/kg, 500 ug/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 50mg/kg, or 100 mg/kg of proprotein can serve as an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily dosage might range from about 1 ug/kg to 100 mg/kg or more,depending on factors such as those mentioned herein. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful.

The proprotein composition will be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the proprotein, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of a proprotein to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat a disease or disorder.

Generally, alleviation or treatment of a disease or disorder involvesthe lessening of one or more symptoms or medical problems associatedwith the disease or disorder. For example, in the case of cancer, thetherapeutically effective amount of the drug can accomplish one or acombination of the following: reduce the number of cancer cells; reducethe tumor size; inhibit (i.e., to decrease to some extent and/or stop)cancer cell infiltration into peripheral organs; inhibit tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Insome embodiments, a composition of this invention can be used to preventthe onset or reoccurrence of the disease or disorder in a subject ormammal

Proproteins can substantially reduce the known side-effects and improvethe efficacy of know drugs, for example those known drugs listed inTable 1.

Proproteins can be used in combination (e.g., in the same formulation orin separate formulations) with one or more additional therapeutic agentsor treatment methods (“combination therapy”). A proprotein can beadministered in admixture with another therapeutic agent or can beadministered in a separate formulation. Therapeutic agents and/ortreatment methods that can be administered in combination with aproprotein, and which are selected according to the condition to betreated, include surgery (e.g., surgical removal of cancerous tissue),radiation therapy, bone marrow transplantation, chemotherapeutictreatment, certain combinations of the foregoing, and the like.

Exemplary Embodiments

The compositions and proproteins provided here in can be useful for avariety of purposes including therapeutics and diagnostics.

Use of Proproteins that Modulate Interferon Signaling Pathways in theTreatment of Liver Conditions

Where the proprotein contains a functional protein that modulatesinterferon signaling, for example when the functional protein is IFN-α,the proprotein finds use in treatment of conditions such as Hepatitis Cviral infection and liver cancers (for e.g. hepatocellular cancer).

An IFN-α proprotein can be used as a therapeutic and/or diagnosticagent. Such a proprotein would be activatable by a cleaving agent (e.g.,enzyme, such as a matriptase, HCV-NS3/4, plasmin or other enzyme asdiscussed herein) which co-localizes at the liver. Exemplary proproteinsfor the treatment of Hepatitis C infection are Matriptase-activatedpro-IFN-α and HCV-NS3/4-activated pro-IFN-α.

An exemplary proprotein useful for the treatment and/or diagnosis ofHepatitis C infection can be a PEGylated pro-interferon alfa-2a or anenzyme-activatable masked PEGylated interferon alfa-2a, such as aproprotein form of PEGASYS® or an enzyme-activatable masked PEGASYS®.For example, the proprotein can be Matriptase or HCV NS3/4 activatable.Other exemplary proteins available for use in interferon-relatedproprotein compositions are presented in Table 1.

Cancer Inhibiting Proproteins

Cancer inhibiting proproteins find use in treatment of several types oftumors.

Where the proprotein contains a functional protein that modulates theNotch pathway, the proprotein finds use in treatment of conditions suchas cancers, for example breast cancer and prostate cancer. In oneembodiment the proprotein can contain an enzyme-activatable solubleNotch receptor or Notch receptor fragment. Exemplary enzyme-activatableNotch containing proproteins for the treatment of various cancersinclude but are not limited to a legumain-activatable pro-Notch1 for thetreatment of colorectal cancer, legumain-activatable pro-Notch1 for thetreatment of head and neck cancer, legumain-activatable pro-Notch1 forthe treatment of pancreatic cancer, legumain-activatable pro-Notch1 forthe treatment of lung cancer, legumain-activatable pro-Notch1 for thetreatment of ovarian cancer, PSA-activatable pro-Notch1 for thetreatment of prostate cancer, plasmin-activatable pro-Notch1 for thetreatment of triple negative breast cancer, plasmin-activatablepro-Notch1 for the treatment of colorectal cancer, plasmin-activatablepro-Notch1 for the treatment of head and neck cancer,plasmin-activatable pro-Notch1 for the treatment of pancreatic cancer,plasmin-activatable pro-Notch1 for the treatment of lung cancer,plasmin-activatable pro-Notch1 for the treatment of ovarian cancer,uPA-activatable pro-Notch1 for the treatment of triple negative breastcancer, uPA-activatable pro-Notch1 for the treatment of colorectalcancer, uPA-activatable pro-Notch1 for the treatment of head and neckcancer, uPA-activatable pro-Notch1 for the treatment of pancreaticcancer, uPA-activatable pro-Notch1 for the treatment of lung cancer, ora uPA-activatable pro-Notch1 for the treatment of ovarian cancer.

Angiogenesis inhibiting proproteins find use in treatment of solidtumors in a subject (e.g., human), particularly those solid tumors thathave an associated vascular bed that feeds the tumor such thatinhibition of angiogenesis can provide for inhibition or tumor growth.Anti-angiogenesis proproteins also find use in other conditions havingone or more symptoms amenable to therapy by inhibition of abnormalangiogenesis.

In general, abnormal angiogenesis occurs when new blood vessels eithergrow excessively, insufficiently or inappropriately (e.g., the location,timing or onset of the angiogenesis being undesired from a medicalstandpoint) in a diseased state or such that it causes a diseased state.Excessive, inappropriate or uncontrolled angiogenesis occurs when thereis new blood vessel growth that contributes to the worsening of thediseased state or causes a diseased state, such as in cancer, especiallyvascularized solid tumors and metastatic tumors (including colon, lungcancer (especially small-cell lung cancer), or prostate cancer),diseases caused by ocular neovascularization, especially diabeticblindness, retinopathies, primarily diabetic retinopathy or age-inducedmacular degeneration and rubeosis; psoriasis, psoriatic arthritis,haemangioblastoma such as haemangioma; inflammatory renal diseases, suchas glomerulonephritis, especially mesangioproliferativeglomerulonephritis, haemolytic uremic syndrome, diabetic nephropathy orhypertensive neplirosclerosis; various imflammatory diseases, such asarthritis, especially rheumatoid arthritis, inflammatory bowel disease,psorsasis, sarcoidosis, arterial arteriosclerosis and diseases occurringafter transplants, endometriosis or chronic asthma and other conditionsthat will be readily recognized by the ordinarily skilled artisan. Thenew blood vessels can feed the diseased tissues, destroy normal tissues,and in the case of cancer, the new vessels can allow tumor cells toescape into the circulation and lodge in other organs (tumormetastases).

Proprotein-based anti-angiogenesis therapies can also find use intreatment of graft rejection, lung inflammation, nephrotic syndrome,preeclampsia, pericardial effusion, such as that associated withpericarditis, and pleural effusion, diseases and disorders characterizedby undesirable vascular permeability, e.g., edema associated with braintumors, ascites associated with malignancies, Meigs' syndrome, lunginflammation, nephrotic syndrome, pericardial effusion, pleuraleffusion, permeability associated with cardiovascular diseases such asthe condition following myocardial infarctions and strokes and the like.

Other angiogenesis-dependent diseases that may be treated usinganti-angiogenic proproteins as described herein include angiofibroma(abnormal blood of vessels which are prone to bleeding), neovascularglaucoma (growth of blood vessels in the eye), arteriovenousmalformations (abnormal communication between arteries and veins),nonunion fractures (fractures that will not heal), atheroscleroticplaques (hardening of the arteries), pyogenic granuloma (common skinlesion composed of blood vessels), scleroderma (a form of connectivetissue disease), hemangioma (tumor composed of blood vessels), trachoma(leading cause of blindness in the third world), hemophilic joints,vascular adhesions and hypertrophic scars (abnormal scar formation).

Amounts of proproteins for administration to provide a desiredtherapeutic effect will vary according to a number of factors such asthose discussed above. In general, in the context of cancer therapy, atherapeutically effective amount of a proprotein is an amount that thatis effective to inhibit angiogenesis, and thereby facilitate reductionof, for example, tumor load, atherosclerosis, in a subject by at leastabout 5%, at least about 10%, at least about 20%, at least about 25%, atleast about 50%, at least about 75%, at least about 85%, or at leastabout 90%, up to total eradication of the tumor, when compared to asuitable control. In an experimental animal system, a suitable controlmay be a genetically identical animal not treated with the agent. Innon-experimental systems, a suitable control may be the tumor loadpresent before administering the agent. Other suitable controls may be aplacebo control.

Whether a tumor load has been decreased can be determined using anyknown method, including, but not limited to, measuring solid tumor mass;counting the number of tumor cells using cytological assays;fluorescence-activated cell sorting (e.g., using antibody specific for atumor-associated antigen) to determine the number of cells bearing agiven tumor antigen; computed tomography scanning, magnetic resonanceimaging, and/or x-ray imaging of the tumor to estimate and/or monitortumor size; measuring the amount of tumor-associated antigen in abiological sample, e.g., blood or serum; and the like.

In some embodiments, the methods are effective to reduce the growth rateof a tumor by at least about 5%, at least about 10%, at least about 20%,at least about 25%, at least about 50%, at least about 75%, at leastabout 85%, or at least about 90%, up to total inhibition of growth ofthe tumor, when compared to a suitable control. Thus, in theseembodiments, “effective amounts” of a proprotein are amounts that aresufficient to reduce tumor growth rate by at least about 5%, at leastabout 10%, at least about 20%, at least about 25%, at least about 50%,at least about 75%, at least about 85%, or at least about 90%, up tototal inhibition of tumor growth, when compared to a suitable control.In an experimental animal system, a suitable control may be tumor growthrate in a genetically identical animal not treated with the agent. Innon-experimental systems, a suitable control may be the tumor load ortumor growth rate present before administering the agent. Other suitablecontrols may be a placebo control.

Whether growth of a tumor is inhibited can be determined using any knownmethod, including, but not limited to, an in vivo assay for tumorgrowth; an in vitro proliferation assay; a 3H-thymidine uptake assay;and the like.

Biodistribution Considerations

The therapeutic potential of the compositions described herein allow forgreater biodistribution and bioavailability of the modified functionalprotein. The compositions described herein provide a protein therapeutichaving an improved bioavailability wherein the affinity of binding ofthe functional protein therapeutic to its binding partner is lower in ahealthy tissue when compared to a diseased tissue. A pharmaceuticalcomposition comprising a functional protein coupled to a peptide maskcan display greater affinity to its binding partner in a diseased tissuethan in a healthy tissue. In preferred embodiments, the affinity in thediseased tissue is 5-10,000,000 times greater than the affinity in thehealthy tissue. In an exemplary embodiment, the affinity in the diseasedtissue is about 10,000 times greater than the affinity in the healthytissue.

Generally stated, the present disclosure provides for a proproteintherapeutic having an improved bioavailability wherein the affinity ofbinding of the therapeutic to its binding partner is lower in a firsttissue when compared to the binding of the therapeutic to its bindingpartner in a second tissue. By way of example in various embodiments,the first tissue is a healthy tissue and the second tissue is a diseasedtissue; the first tissue is an early stage tumor and the second tissueis a late stage tumor; the first tissue is a benign tumor and the secondtissue is a malignant tumor; the first tissue is liver tissue and thesecond tissue is non liver tissue; the first tissue is uninfected livertissue and the second tissue is virally infected liver tissue; or thefirst tissue and second tissues are spatially separated. In the specificexample where the first tissue is a healthy tissue and the second tissueis a diseased tissue, the diseased tissue can be a tumor-containingtissue, an inflamed tissue, or a viral infected tissue. In anotherspecific example, the first tissue is epithelial tissue and the secondtissue is breast, head, neck, lung, pancreatic, nervous system, liver,prostate, urogenital, or cervical tissue.

In one exemplary embodiment, the invention provides for a proproteintherapeutic for the treatment of Hepatitis C having an improvedbioavailability. Such a proprotein contains a functional protein coupledto a peptide mask and a cleavable linker, wherein the affinity ofbinding of the functional protein therapeutic to its target is higher inliver tissue when compared to the binding of the functional proteintherapeutic to its target in a non-liver tissue, wherein target ispresent in both tissues. Furthermore, the proprotein can contain acleavable linker comprising a substrate specific for an enzymeupregulated in Hepatitis C or a hepatocellular cancer affected tissue,for example a substrate for a matriptase or HCV NS3/4 enzyme.

Pharmaceutical Compositions

Proproteins of the present disclosure can be incorporated intopharmaceutical compositions containing, for example, a therapeuticallyeffective amount of an activatable masked protein of interest and acarrier pharmaceutically acceptable excipient (also referred to as apharmaceutically acceptable carrier). Many pharmaceutically acceptableexcipients are known in the art, are generally selected according to theroute of administration, the condition to be treated, and other suchvariables that are well understood in the art. Pharmaceuticallyacceptable excipients have been amply described in a variety ofpublications, including, for example, A. Gennaro (2000) “Remington: TheScience and Practice of Pharmacy,” 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; andHandbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds.,3rd ed. Amer. Pharmaceutical Assoc. Pharmaceutical compositions can alsoinclude other components such as pH adjusting and buffering agents,tonicity adjusting agents, stabilizers, wetting agents and the like. Insome embodiments, nanoparticles or liposomes carry a pharmaceuticalcomposition comprising a proprotein.

Suitable components for pharmaceutical compositions of proproteins canbe guided by pharmaceutical compositions that may be available for thefunctional protein to be masked.

In general, pharmaceutical formulations of one or more proproteins areprepared for storage by mixing the proprotein having a desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes. Pharmaceutical formulations may also contain more than oneactive compound as necessary for the particular indication beingtreated, where the additional active compounds generally are those withactivities complementary to the proprotein.

The pharmaceutical formulation can be provided in a variety of dosageforms such as a systemically or local injectable preparation. Thecomponents can be provided in a carrier such as a microcapsule, e.g.,such as that prepared by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations are also within the scope ofproprotein-containing formulations. Exemplary sustained-releasepreparations can include semipermeable matrices of solid hydrophobicpolymers containing the antibody, which matrices are in the form ofshaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andγ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

Proproteins can be conjugated to delivery vehicles for targeted deliveryof an active agent that serves a therapeutic purpose. For example,proproteins can be conjugated to nanoparticles or liposomes having drugsencapsulated therein or associated therewith. In this manner, specific,targeted delivery of the drug can be achieved. Methods of linkingpolypeptides to liposomes are well known in the art and such methods canbe applied to link proproteins to liposomes for targeted and orselective delivery of liposome contents. By way of example, polypeptidescan be covalently linked to liposomes through thioether bonds. PEGylatedgelatin nanoparticles and PEGylated liposomes have also been used as asupport for the attachment of polypeptides, e.g., single chainantibodies. See, e.g., Immordino et al. (2006) Int J. Nanomedicine.September; 1(3): 297-315, incorporated by reference herein for itsdisclosure of methods of conjugating polypeptides, e.g., antibodyfragments, to liposomes.

In certain embodiments the proproteins of the present are furtherconjugated to protective chains such as PEG or mPEG, or any alkyl-PEG.Such conjugates would be less susceptible to non specific in vivohydrolytic cleavage, have enhanced in vivo half life, and reduce theimmunogenicity of the functional protein while maintaining biologicalactivity.

Non-Therapeutic Uses of Proproteins

Proproteins can also be used in diagnostic and/or imaging methods. Forexample, proproteins having an enzymatically cleavable linker can beused to detect the presence or absence of an enzyme that is capable ofcleaving the cleavable linker. Such proproteins can be used indiagnostics, which can include in vivo detection (e.g., qualitative orquantitative) of enzyme activity accompanied by presence of a bindingpartner of interest through measured accumulation of activatedproproteins in a given tissue of a given host organism.

For example, the cleavable linker can be selected to be an enzymesubstrate for an enzyme found at the site of a tumor, at the site of aviral or bacterial infection at a biologically confined site (e.g., suchas in an abscess, in an organ, and the like). Using methods familiar toone skilled in the art, a detectable label (e.g., a fluorescent label)can be conjugated to the functional protein or other region of theproprotein. Using a functional protein specific to a disease target,along with an enzyme whose activity is elevated in the disease tissue ofinterest, proproteins can exhibit increased rate of binding to diseasetissue relative to tissues where the cleavable linker-specific enzyme isnot present at a detectable level or is present at a lower level than indisease tissue. Because the enzyme specific for the cleavable linker isnot present at a detectable level (or is present at lower levels) innon-diseased tissues, accumulation of activated proprotein in thediseased tissue is enhanced relative to non-disease tissues.

Non-limiting examples of detectable labels that can be used asdiagnostic agents include imaging agents containing radioisotopes suchas indium or technetium; contrasting agents for MRI and otherapplications containing iodine, gadolinium or iron oxide; enzymes suchas horse radish peroxidase, alkaline phosphatase, or β-galactosidase;fluorescent substances and fluorophores such as GFP, europiumderivatives; luminescent substances such as N-methylacrydium derivativesor the like.

EXAMPLES Example 1 Screening of a Peptide Library and Identification ofPeptide Masks Specific for IFN-α

In order to identify peptide masks for Interferon-α (IFN-α), a peptidelibrary was screened. IFN-α was used to screen a random 15× peptidelibrary, where X is any amino acid, with a total diversity of 5×10¹⁰.The screening consisted of an initial round of MACS (magnetic activatedcell sorting) followed by four rounds of FACS (fluorescence activatedcell sorting). The initial MACS and three rounds of FACS were done withbiotinylated IFN-α at a concentration of 500 nM. For MACS, approximately1×10¹¹ cells were screened for binding and 3.4×10⁷ cells were collected.NeutrAvidin-PE was used as a fluorescent probe for the initial FACSrounds. The fourth round of FACS selections was done with 500 nM Dylightlabeled IFN-α (Dylight-IFN-α). The third and fourth round of FACSsorting is shown labeled with Dylight-IFN-α in FIG. 2.

Exemplary binding peptides are shown in Table 3 below.

TABLE 3 IFN-α Binding peptides 47 IAYLEYYEHLHMAYG 49 TDVDYYREWCWTQVS49CS TDVDYYREWSWTQVS

Example 2 Construction and Expression of Pro-IFN-α

Construction of Interferon-α under PhoA Control: The human Interferon-αgene was purchased from Open Biosystems. IFN-α was cloned into thePhagmid X (PhoA driven bacterial expression vector) in the followingmanner. IFN-α was amplified using primers CX0573 and CX0566. The PhoApromoter was amplified from the Phagmid X using the primers CX0571 andCX0572. These two overlapping products were combined into one polymerasechain reaction and amplified using the primers CX0581 and CX0572. Thefinal product was cloned into Phagmid X using the HindIII and EcoRIrestriction sites.

Construction of Masked Interferon-α under PhoA Control: A mask acceptingvector with GGS linker and no protease substrate was constructed asfollows. The overlapping forward primers CX0577, CX0579, and CX0580 wereused with the reverse primer CX0566 to amplify the IFN-α cDNA with a GGSlinker and mask accepting site. This product was cloned into the STIIcontaining Phagmid X vector using the BamHI and EcoRI restriction sites.This vector was then used as a template for the construction of theMMP-9 substrate containing vector. Two overlapping PCR products wereamplified using the primer pair CX0573/CX0612 and CX0611/CX0566. Thesetwo products were combined into a PCR, amplified with the primers CX0573and CX0566, and cloned into the Phagmid X using the HindIII and EcoRIrestriction sites.

The IFN-α peptide masks were cloned into the MMP-9 Pro-protein vectorusing the SfiI and XhoI sites. The 47 and 49 peptide masks (Table 3)were then amplified using CX0289/CX0448 and CX0582/CX0583, respectively,using the ecpX3.0 clones that encoded the bacterial displayed maskingpeptide indicated. The CX0582/CX0583 primer pair mutated the Cys in the49 masking peptide to a Ser creating the masking peptide 49CS (Table 3).

TABLE 4 Primer Sequences for Construction of Masked IFN-α CX0289gctttcaccgcaggtacttccgtagctggccagtctggcc CX0448gagttttgtcggatccaccagagccaccgctgccaccgctcgagcc CX0566gcgttatcccgaattcctagtggtgatggtgatgatgttccttacttcttaaactttcttgc CX0571agtgaattgtaagctttggagattatcgtcac CX0572caggctgtgggtttgaggcagatcacacattttattttctccatgtacaaatac CX0573tgtgatctgcctcaaacccacagcctg CX0577 ggtggcagcatgtgtgatctgcctcaaacccacCX0579 ggctcgagcggcggctccggcggtagcggtggctctggtggcagcatgtgtgatctgc CX0580tgcgtatgcaggatccggccagtctggccagcaagtcattctgagaagcggctcgagcggcggctccCX0582 ttccgtagctggccagtctggccagacggacgtggactattatagggagtggtc CX0583gctgccaccgctcgagcctgatacttgagtccaggaccactccctataatagtc CX0611catgccactgggcttcctgggtccgggtggcagcatgtgtgatc CX0612ccaggaagcccagtggcatgtgcacggagccgccgctcgagccgc

Interferon-α expression and inclusion body purification: Interferon andpro-Interferon-α constructs were expressed in the cytoplasm of E. coliunder control of the PhoA promoter. Inclusion bodies were purified asfollows: bacteria from 1 Liter of fresh overnight culture were grown inphosphate limiting media (per Liter=3.57 g (NH₄)₂SO₄, 0.71 g Nacitrate-2H₂O, 1.07 g KCl, 5.36 g Yeast Extract, 5.36 gHycaseSF-Sheffield, pH adjusted to 7.3 with KOH, volume adjusted to 872ml, autoclaved. Supplemented post-autoclave with 110 peptide mask MOPSpH7.3, 0.5% glucose, 7 uM MgSO₄ and 50 ug/ml carbenicillin). The culturewas pelleted and then lysed with 20 mL of BPER11 (Pierce). The lysatewas centrifuged at 14,000×g and the supernatant discarded. The pelletwas then resuspended in a 1:10 BPER11 to water solution, 720 Ku oflysozyme and 40 Ku of DNAseI were added, and lysate was incubated atroom temperature for 1 hr. The lysate was centrifuged at 14,000×g andthe inclusion bodies (IBs) were washed an additional time in 1:20BPER11. Pelleted inclusion bodies were stored at −20° C. until furtheruse.

Interferon-α purification and refolding: Inclusion bodies isolated from1 Liter of culture were solubilized in 20 mL of IB solubilization buffer(50 peptide mask Tris, 8 M Urea, 1 peptide mask TCEP, pH 8.0). Insolubleprotein was removed by centrifugation before adding the solubilizedprotein to a Ni-NTA column (Qiagen). The bound protein was washed with 5mL of IB solubilization buffer followed by 5 mL of IB solubilizationbuffer with 5 peptide mask β-mercaptoethanol instead of TCEP. Purifiedprotein was eluted with Elution Buffer (0.2M Glycine, 8M Urea, pH 3.0)and added in a drop-wise fashion to 100 mL of stirring chilled RefoldingBuffer (0.75 M Arginine, 0.055% PEG (w/v), 2.2 mM CaCl₂, 2.2 mM MgCl₂,55 mM Tris, 0.44 mM KCL, 10.56M NaCl, 4 mM reduced glutathione, 0.4 mMoxidized glutathione, pH 7.5). Refolding was allowed to proceedovernight at 4° C. with constant slow stirring. Following refolding, theprotein was dialyzed extensively into PBS before being applied to aNi-NTA column. Bound protein was washed with PBS and Eluted withImidizole Elution Buffer (50 mM Tris, 300 mM NaCl, 250 mM Imidizole).Purified protein was concentrated and buffer exchanged to PBS, pH 7.4using an Amicon Centrifuge concentrator.

Example 3 Analysis of Pro-IFN-α Masking and Unmasking

To demonstrate masking of the Pro-IFN-α, the refolded proteins,47-MMP-IFN-α or 49-MMP-IFN-α were diluted 1:1 in MMP-9 digestion buffer(50 mM Tris, 20 mM NaCl, 2 mM CaCl₂, 100 μM ZnCl₂, pH 6.82) and half ofthe sample was digested with about 35 Units of MMP-9 for 3 hrs at 37° C.Subsequently, 60, 40, 20, and 6.6 μL of the digested and undigestedmaterial was added to 400 μL of 2% non-fat dry milk in PBS-T (PBS, 0.05%TWEEN, pH 7.4) and analyzed by ELISA, as described:

Interferon ELISA's: A recombinant Interferon receptor 1-Fc (IFNR1-Fc)fusion protein (R & D Systems) was used to detect IFN-α binding.Briefly, the receptor was absorbed to ELISA plates at a concentration of5 μg/mL in PBS for 1 hr at RT. Wells were then blocked with 2% non-fatdry milk in PBS-T for 1 hr at RT. Interferon-α was added at threeconcentrations, 60, 40, 20 and 6.6 nM, to the wells in 100 μL of 2%non-fat dry milk in PBS-T. Wells were washed 3 times with PBS-T and theinterferon was detected with an anti-His₆ monoclonal antibody(Invitrogen) at a titer of 1:1000 mixed with an anti-muFc-HRP conjugate(Fisher) at a titer of 1:2000 in a 100 uL of 2% non-fat dry milk inPBS-T per well. The ELISA was developed with 100 μL of TMB (Pierce)following the manufacturer's protocol (FIG. 3). FIG. 3 shows the bindingof two Pro-Interferon-α molecules, Pro-Interferon-α-47 (Tables 7 and 8)and Pro-Interferon-α-49CS (Tables 8 and 9), before and after treatmentwith MMP-9. The first four bars of FIG. 3 (small checked) show thatbefore treatment Pro-Interferon-α-49CS cannot bind to IFNRA, howeverafter MMP-9 removal of Mask 49CS the resulting IFN-α (second set of fourbars, Figure, large checked) molecule binds to IFNRA. In contrast Mask47 weakly blocks IFN-α binding to IFNRA when incorporated intoPro-Interferon-α-47 (FIG. 3, third set of bars, horizontal lines) whichis restored by treatment with MMP9 (FIG. 3, final four bars, verticallines).

TABLE 5 Nucleotide Sequence of Interferon-αatgtgtgatctgcctcaaacccacagcctgggtagcaggaggaccttgatgctcctggcacagatgaggagaatctctcttttctcctgcttgaaggacagacatgactttggatttccccaggaggagtttggcaaccagttccaaaaggctgaaaccatccctgtcctccatgagatgatccagcagatcttcaatctcttcagcacaaaggactcatctgctgcttgggatgagaccctcctagacaaattctacactgaactctaccagcagctgaatgacctggaagcctgtgtgatacagggggtgggggtgacagagactcccctgatgaaggaggactccattctggctgtgaggaaatacttccaaagaatcactctctatctgaaagagaagaaatacagcccttgtgcctgggaggttgtcagagcagaaatcatgagatctttttctttgtcaacaaacttgcaagaaagtttaagaagtaaggaacatcaccatcatcaccat

TABLE 6 Amino Acid Sequence of Interferon-α: Parentheses delineate thedemarcations between the various sequence domains: (IFN-α) -- (affinitytag) (MCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE)(HHHHHH)

TABLE 7 Nucleotide Sequence of Pro-Interferon-α -- 47ggccagtctggccagattgcgtaccttgagtattatgagcacctacatatggcctacggctcgagcggcggctccgtgcacatgccactgggcttcctgggtccgggtggcagcatgtgtgatctgcctcaaacccacagcctgggtagcaggaggaccttgatgctcctggcacagatgaggagaatctctcttttctcctgcttgaaggacagacatgactttggatttccccaggaggagtttggcaaccagttccaaaaggctgaaaccatccctgtcctccatgagatgatccagcagatcttcaatctcttcagcacaaaggactcatctgctgcttgggatgagaccctcctagacaaattctacactgaactctaccagcagctgaatgacctggaagcctgtgtgatacagggggtgggggtgacagagactcccctgatgaaggaggactccattctggctgtgaggaaatacttccaaagaatcactctctatctgaaagagaagaaatacagcccttgtgcctgggaggttgtcagagcagaaatcatgagatctttttctttgtcaacaaacttgcaagaaagtttaagaagtaaggaacatcaccatcatcaccat

TABLE 8 Amino Acid Sequence of Pro-Interferon-α - 47 Parenthesesdelineate the demarcations between the various sequence domains:(Linker) -- (Masking Peptide) -- (Linker) -- (MMP-9 substrate) --(Linker) -- (IFN-α) -- (Affinity tag)(GQSGQ)(IAYLEYYEHLHMAY)(GSSGGS)(VHMPLGFLGP)(GGS)(MCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE)(HHHHHH)

TABLE 9 Nucleotide Sequence of Pro-Interferon-α - 49CSggccagtctggccagacggacgtggactattatagggagtggtcctggactcaagtatcaggctcgagcggcggctccgtgcacatgccactgggcttcctgggtccgggtggcagcatgtgtgatctgcctcaaacccacagcctgggtagcaggaggaccttgatgctcctgcacagatgaggagaatcttctcttttctcctgcttgaaggacagacatgactttggatttccccaggaggagtttggcaaccagttccaaaaggctgaaaccatccctgtcctccatgagatgatccagcagatcttcaatctcttcagcacaaaggactcatctgctgcttgggatgagaccctcctagacaaattctacactgaactctaccagcagctgaatgacctggaagcctgtgtgatacagggggtgggggtgacagagactcccctgatgaaggaggactccattctggctgtgaggaaatacttccaaagaatcactctctatctgaaagagaagaaatacagcccttgtgcctgggaggttgtcagagcagaaatcatgagatctttttctttgtcaacaaacttgcaagaaagtttaagaagtaaggaacatcaccatcatcaccat

TABLE 10 Amino Acid Sequence of Pro-Interferon-α - 49CS Parenthesesdelineate the demarcations between the various sequence domains:(Linker) -- (Masking Peptide) -- (Linker) -- (MMP-9 substrate) --(Linker) -- (IFN-α) -- (Affinity tag)(GQSGQ)(TDVDYYREWSWTQVS)(GSSGGS)(VHMPLGFLGP)(GGS)(MCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE)(HHHHHH)

Example 4 Construction and Testing of a Matriptase or HCV NS3/4Activatable IFN-α Proprotein Library Displaying Candidate Substrates andPeptide Masks

In order to identify IFN-α proproteins having desired activatingcharacteristics (i.e., decreased binding to its IFNRA receptor when inan uncleaved conformation relative to IFNRA receptor binding when in acleaved conformation), candidate IFN-α proproteins having variablematriptase or HCV NS3/4 cleavable linkers and different variable aminoacid sequences in the peptide masks and varying positions of thecysteine in the peptide mask were generated.

Consensus sequences for Matriptase and HCV NS3/4 are provided here inTables 11-12.

TABLE 11 Matriptase Consensus Sequences XXQAR(A/V)X AGPR

TABLE 12 HCV NS3/4 Consensus Sequences DEXXXC(A/S) DLXXXT(A/S)

Interferon-α purification and refolding: Inclusion bodies isolated from1 Liter of culture were solubilized in 20 mL of IB solubilization buffer(50 peptide mask Tris, 8 M Urea, 1 peptide mask TCEP, pH 8.0). Insolubleprotein was removed by centrifugation before adding the solubilizedprotein to a Ni-NTA column (Qiagen). The bound protein was washed with 5mL of IB solubilization buffer followed by 5 mL of IB solubilizationbuffer with 5 peptide mask β-mercaptoethanol instead of TCEP. Purifiedprotein was eluted with Elution Buffer (0.2M Glycine, 8M Urea, pH 3.0)and added in a drop-wise fashion to 100 mL of stirring chilled RefoldingBuffer (0.75 M Arginine, 0.055% PEG (w/v), 2.2 mM CaCl₂, 2.2 mM MgCl₂,55 mM Tris, 0.44 mM KCL, 10.56M NaCl, 4 mM reduced glutathione, 0.4 mMoxidized glutathione, pH 7.5). Refolding was allowed to proceedovernight at 4° C. with constant slow stirring. Following refolding, theprotein was dialyzed extensively into PBS before being applied to aNi-NTA column. Bound protein was washed with PBS and Eluted withImidizole Elution Buffer (50 mM Tris, 300 mM NaCl, 250 mM Imidizole).Purified protein was concentrated and buffer exchanged to PBS, pH 7.4using an Amicon Centrifuge concentrator.

To demonstrate masking of the Pro-IFN-α, the refolded proteins,Mask-Matriptase-IFN-α or Mask-HCV NS3/4-IFN-α were diluted 1:1 indigestion buffer (50 mM Tris, 20 mM NaCl, 2 mM CaCl₂, pH 7.2) and halfof the sample was digested with about 20 nM of Matriptase or HCV NS3/4for 3 hrs at 37° C. Subsequently, 60, 40, 20, and 6.6 μL of the digestedand undigested material was added to 400 μL of 2% non-fat dry milk inPBS-T (PBS, 0.05% TWEEN, pH 7.4) and analyzed by ELISA, as describedbelow.

Interferon ELISA's: A recombinant Interferon receptor 1-Fc (IFNR1-Fc)fusion protein (R & D Systems) was used to detect IFN-α binding.Briefly, the receptor was absorbed to ELISA plates at a concentration of5 μg/mL in PBS for 1 hr at RT. Wells were then blocked with 2% non-fatdry milk in PBS-T for 1 hr at RT. Interferon-α was added to the well in100 μL of 2% non-fat dry milk in PBS-T. Wells were washed 3 times withPBS-T and the interferon was detected with an anti-His₆ monoclonalantibody (Invitrogen) at a titer of 1:1000 mixed with an anti-muFc-HRPconjugate (Fisher) at a titer of 1:2000 in a 100 uL of 2% non-fat drymilk in PBS-T per well. The ELISA was developed with 100 μL of TMB(Pierce) following the manufacturer's protocol.

IFN-α masking efficiency assay: IFNR-α is adsorbed to the wells of anELISA plate overnight at about 4° C. The plate is blocked by addition ofabout 150 ul 2% non-fat dry milk (NFDM) in PBS, about 0.5% V/V tween 20(PBST), and incubated at room temperature for about 1 hour. The plate iswashed about three times with PBST. About 50 ul superblock (ThermoScientific) supplemented with protease inhibitors (Complete, Roche) isadded. About 50 ul of a solution of pro-IFN-α dissolved in superblockwith protease inhibitors (Complete, Roche) is added and incubated atabout 37° C. for desired time. The plate is washed about three timeswith PBST. About 100 ul of anti-His-HRP in 2% NFDM/PBST is added andincubated at room temperature for about 1 hour. The plate is washedabout four times with PBST and about twice with PBS. The assay isdeveloped using TMB (Thermo Scientific) as per manufacturer'sdirections. An efficiently masked pro-IFN-α would be expected to showless than 10% of the binding observed for unmasked IFN-α.

Example 5 Construction of a Masked Soluble Plasmin or MMP-9 ActivatableNotch Receptor Protein

Sequences to construct a masked plasmin-activatable soluble NotchReceptor fragment and a masked MMP9-activatable soluble Notch Receptorfragment are provided in this example. These proproteins are inactiveunder normal conditions due to the attached peptide mask. Bacterial cellsurface display is used to find suitable peptide masks for the solubleNotch receptor protein. In this example, selected peptide masks arecombined with either a plasmin or MMP-9 enzyme substrate to be used as atrigger to create a proprotein construct that becomes competent fortargeted binding after enzyme-mediated activation.

The gene encoding human Notch1 EGF-like domains 11-13 (hN1₁₁₋₁₃) wasconstructed by PCR assembly of overlapping oligonucleotides CX509-CX528(Table 13), digested with EcoRI/BglII, and ligated to pINFUSE-hIgG1-Fc2(InvivoGen) that had been digested with EcoRI/BglII. The resultingplasmid was used for CHO—S expression of hN1₁₁₋₁₃ fused to the Fc domainof human IgG1 (hN1₁₁₋₁₃-hFc). The hN1₁₁₋₁₃-hFc was purified from cellculture supernatant by Protein A chromatography and labeled withPEG-biotin or DyLight488 (Thermo Pierce) following standard protocols.

TABLE 13 Oligonucleotides used for constructing hN111-13 CX509GTCACGAATTCGCAGGACGTCGACGAGTGCTCGCTGGGT CX510GCTCGCAGGGGTTGGCACCCAGCGAGCACTCGT CX511GCCAACCCCTGCGAGCATGCGGGCAAGTGCATCA CX512GAAGGAGCCCAGCGTGTTGATGCACTTGCCCGCAT CX513ACACGCTGGGCTCCTTCGAGTGCCAGTGTCTGCAGG CX514CGGGGGCCCGTGTAGCCCTGCAGACACTGGCACTC CX515GCTACACGGGCCCCCGATGCGAGATCGACGTCAACG CX516ACGGGTTCGAGACGCACTCGTTGACGTCGATCTCGCAT CX517AGTGCGTCTCGAACCCGTGCCAGAACGACGCCACC CX518CCCAATCTGGTCCAGGCAGGTGGCGTCGTTCTGGC CX519TGCCTGGACCAGATTGGGGAGTTCCAGTGCATCTGCATGC CX520CACACCCTCGTAGCCGGGCATGCAGATGCACTGGAACTC CX521CCGGCTACGAGGGTGTGCACTGCGAGGTCAACACAGA CX522GGCTGCTGGCACACTCGTCTGTGTTGACCTCGCAGTG CX523CGAGTGTGCCAGCAGCCCCTGCCTGCACAATGGCC CX524TCATTGATCTTGTCCAGGCAGCGGCCATTGTGCAGGCAGG CX525GCTGCCTGGACAAGATCAATGAGTTCCAGTGCGAGTGCCC CX526GCCCAGTGAAGCCCGTGGGGCACTCGCACTGGAAC CX527CACGGGCTTCACTGGGCATCTGTGCCAGGGCAGC CX528GTCGTCTGGTGGATCCACCGCTGCCCTGGCACAGAT

A library of peptides containing 15 random amino acids displayed on theE. coli surface was used for screening for peptides that bindhN1₁₁₋₁₃-hFc. Approximately 1.5×10¹¹ library cells, induced with 0.04%arabinose for 45 minutes at 37° C., were depleted of streptavidin (SA)binders by incubating with 10⁹ SA-coated magnetic beads (InvitrogenDynabeads MyOne SA-C1) in Tris-buffered saline (50 mM Tris-HCl ph 7.4,150 mM NaCl) with 2 mM CaCl₂ and 0.5% bovine serum albumin (TBS—Ca—B).The magnetic beads were then removed using a magnet, and the remainingcell population was mixed with 300 nM hN1₁₁₋₁₃-hFc that had beenbiotinylated with NHS-PEG-biotin (Thermo Pierce) (hN1₁₁₋₁₃-hFc-biot) and5 μM pooled human IgG that had been depleted of E. coli-bindingantibodies (hIgG). The cells were washed with TBS—Ca—B, and incubatedwith 10⁹ SA-coated beads and 5 μM hIgG. The beads were then washed threetimes, and incubated in LB medium overnight to amplify thehN1₁₁₋₁₃-hFc-binding population. A second round of magnetic selectionwas performed as in the first round, starting with 3×10⁸ cells from thefirst round enriched population, 600 nM hN1₁₁₋₁₃-hFc-biot, 10 μM hIgG,and 5×10⁸ SA-coated beads.

Following two rounds of magnetic selection, the remaining rounds ofscreening were performed on a Becton Dickinson FACSAria flow cytometer.In the first round of FACS, induced cells were incubated with 500 nMhN1₁₁₋₁₃-hFc-biot, 10 μM hIgG in TBS—Ca—B, washed, and incubated withfluorescent secondary label neutravidin-phycoerythrin (NAPE)(Invitrogen) at 10 nM, before sorting by flow cytometry forfluorescently labeled cells. Cells amplified from overnight growth ofthe first round FACS population were induced and subjected to a secondround of sorting with the same labeling conditions as in the first roundor, alternatively, using 50 nM hN1₁₁₋₁₃-hFc-biot. A third round ofsorting was conducted as in the second round but with 100 nMhN1₁₁₋₁₃-hFc-biot and the addition of 27 nM Ypet-Mona-SH3 in thesecondary labeling step. Mona-SH3 binds an epitope on the C-terminus ofthe display scaffold, independent of the random peptide on theN-terminus. Cells were then sorted based on the ratio of 576 nmfluorescence (i.e. NAPE binding) to 530 nm fluorescence (i.e. Ypet-Monabinding) in order to normalize for differences in scaffold display levelon individual cells.

Alternatively, third round sorting was conducted by incubating inducedcells with 10 nM or alternatively, 50 nM unbiotinylated hN1₁₁₋₁₃-hFc inTBS—Ca—B before washing, labeling with fluorescent secondary 20 μg/mlanti-hIgG-DyLight-488, and sorting based on 530 nm fluorescence. Thirdround sorting was also conducted using either 50 nM or 250 nMhN1₁₁₋₁₃-hFc that had been fluorescently labeled with DyLight-488(Thermo Pierce) (hN1₁₁₋₁₃-hFc-Dy488), and 10 μM hIgG, with no secondarylabeling. Colonies derived from FACS round 3 populations enriched forhN1₁₁₋₁₃-hFc binding were used for plasmid sequencing in order todiscover the sequences of the encoded peptides.

Individual clones were tested by flow cytometry for hN1₁₁₋₁₃-hFc bindingby labeling induced cells in TBS—Ca—B with (A.) 50 nM hN1₁₁₋₁₃-hFc-biotor (B.) 100 nM 50 nM hN1₁₁₋₁₃-hFc-biot, followed by 10 nMStreptavidin-R-phycoerythrin (SAPE). Cells were separately labeled with27 nM Ypet-Mona to measure peptide display level. The display scaffoldalone (ecpX3) was used as a negative control. Clones Jag-ecpX3 andRJag-ecpX3 display a fragment of JAG1 and a mutated fragment,respectively, which have been shown to bind Notch1₁₁₋₁₃. (Table 14 andFIG. 4). FIG. 4 shows individual clones that were tested by flowcytometry for hN1₁₁₋₁₃-hFc binding by labeling induced cells in TBS—Ca—Bwith 100 nM hN1₁₁₋₁₃-hFc-biot, followed by 10 nMStreptavidin-R-phycoerythrin (SAPE), and normalized based on the displaylevel of the scaffold. Clone ecpX3 displays the scaffold alone, andclone Jag-ecpX3 displays a peptide derived from Jagged1(RVTCDDYYYGFGCNKFGRPA) that is known to bind Notch1. The clonesresulting from library screening bind hN1₁₁₋₁₃-hFc better than theJagged1-derived peptide.

TABLE 14 Binders to hN1₁₁₋₁₃-hFc after two rounds of magnetic selectionand three rounds of FACS PHB3324 FPLNTFDLVHELLSR PHB3325 FLNDIHRFLHWTDLMPHB3327 PYTFVEQVEYWLHAT PHB3333 ACVIHFLDRISNILE PHB3334 FCYIAAFSAMQRQSCPHB3336 PLYLPEIGWMFGLPT PHB3337 TVLVIPDLHYLYVDR PHB3340 FINNVETALDTIYNLPHB3341 SAKHLHPGRLPPMTK PHB3343 ATMYAYLERLEAILS PHB3349 IYPLDALLRHLNSLCPHB3352 CFPTVVWRELYNLYG PHB3476 NLDFYLNHLYNTLAG PHB3478 DFINSMRSHLQSSDQPHB3479 EPKCSFCSPLIVPSP PHB3480 PNCIESFLSSIHDSL PHB3482 TDNALFLETVQHYLYPHB3485 CYPSISWLFADAPRN PHB3486 ELTQLLNALVDVRNC PHB3487 LLSSFVETMSSILTCPHB3488 YLLRLPSLEELWGPS PHB3489 ATCYIINHWVERYII

TABLE 15 Nucleotide Sequence of the Soluble Notch Receptor Fragmentcaggacgtcgacgagtgctcgctgggtgccaacccctgcgagcatgcgggcaagtgcatcaacacgctgggctccttcgagtgccagtgtctgcagggctacacgggcccccgatgcgagatcgacgtcaacgagtgcgtctcgaacccgtgccagaacgacgccacctgcctggaccagattggggagttccagtgcatctgcatgcccggctacgagggtgtgcactgcgaggtcaacacagacgagtgtgccagcagcccctgcctgcacaatggccgctgcctggacaagatcaatgagttccagtgcgagtgccccacgggcttcactgggcatctgtgccag

TABLE 16 Amino Acid Sequence of the Soluble Notch Receptor Fragmentqdvdecslganpcehagkcintlgsfecqclqgytgprceidvnecvsnpcqndatcldqigefqcicmpgyegvhcevntdecasspclhngrcldkin efqcecptgftghlcg

TABLE 17 Nucleotide Sequence Plasmin Activatable Masked Soluble NotchReceptor Fragment cgcgtaacttgtgacgattactactacggattcgggtgtaacaagtttggtagacccgccggcggcggatcaggcggagggtcaggaggcggtagcggcgggggctccggcggcggttcagggggaggatcccaaggaccaatgttcaaaagcctatgggacggaggccaggacgtcgacgagtgctcgctgggtgccaacccctgcgagcatgcgggcaagtgcatcaacacgctgggctccttcgagtgccagtgtctgcagggctacacgggcccccgatgcgagatcgacgtcaacgagtgcgtctcgaacccgtgccagaacgacgccacctgcctggaccagattggggagttccagtgcatctgcatgcccggctacgagggtgtgcactgcgaggtcaacacagacgagtgtgccagcagcccctgcctgcacaatggccgctgcctggacaagatcaatgagttccagtgcgagtgccccacgggcttcac tgggcatctgtgccag

TABLE 18 Amino Acid Sequence Plasmin Activatable Masked Soluble NotchReceptor Fragment Parentheses delineate the demarcations between thevarious sequence domains: (Peptide Mask) - (Linker) - (PlasminSubstrate) - (GG Linker) - (Soluble Notch Receptor Fragment)(RVTCDDYYYGFGCNKFGRPA)(GGGSGGGSGGGSGGGSGGGSGGGS)(QGPMFKSLWD)(GG)(QDVDECSLGANPCEHAGKCINTLGSFECQCLQGYTGPRCEIDVNECVSNPCQNDATCLDQIGEFQCICMPGYEGVHCEVNTDECASSPCLHNGRCLDKINEFQCECPTGFTGHLCQ)

TABLE 19 Nucleotide Acid Sequence MMP9 Activatable Masked Soluble NotchReceptor Fragment cgcgtaacttgtgacgattactactacggattcgggtgtaacaagtttggtagacccgccggcggcggatcaggcggagggtcaggaggcggtagcggcgggggctccggcggcggttcagggggaggatccgttcatatgcccttgggtttcctggggccaggaggccaggacgtcgacgagtgctcgctgggtgccaacccctgcgagcatgcgggcaagtgcatcaacacgctgggctccttcgagtgccagtgtctgcagggctacacgggcccccgatgcgagatcgacgtcaacgagtgcgtctcgaacccgtgccagaacgacgccacctgcctggaccagattggggagttccagtgcatctgcatgcccggctacgagggtgtgcactgcgaggtcaacacagacgagtgtgccagcagcccctgcctgcacaatggccgctgcctggacaagatcaatgagttccagtgcgagtgccccacgggcttcac tgggcatctgtgccag

TABLE 20 Amino Acid Sequence MMP9 Activatable Masked Soluble NotchReceptor Fragment Parentheses delineate the demarcations between thevarious sequence domains: (Peptide Mask) - (Linker) - (MMP9 Substrate) -(GG Linker) - (Soluble Notch Receptor Fragment)(RVTCDDYYYGFGCNKFGRPA)(GGGSGGGSGGGSGGGSGGGSGGGS)(VHMPLGFLGP)(GG)(QDVDECSLGANPCEHAGKCINTLGSFECQCLQGYTGPRCEIDVNECVSNPCQNDATCLDQIGEFQCICMPGYEGVHCEVNTDECASSPCLHNGRCLDKINEFQCECPTGFTGHLCQ)

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A composition comprising a functional protein that is not an antibodyor an antibody fragment, wherein the functional protein is coupled to apeptide mask that: (i) inhibits binding of the functional protein to itsbinding partner and (ii) does not have an amino acid sequence of thebinding partner.
 2. The composition of claim 1 wherein the functionalprotein is further coupled to a cleavable linker capable of beingcleaved, such that: (i) in an uncleaved state, the peptide mask inhibitsbinding of the functional protein to its binding partner and (ii) in acleaved state, the peptide mask does not inhibit binding of thefunctional protein to its binding partner.
 3. The composition of claim 2wherein the cleavable linker is capable of being specifically cleaved byan enzyme, capable of being reduced by a reducing agent, or capable ofbeing photolysed.
 4. The composition of claim 1 wherein the functionalprotein coupled to a peptide mask is recombinantly expressed.
 5. Thecomposition of claim 1 wherein the peptide mask is unique for thefunctional protein.
 6. The composition of claim 1 wherein the peptidemask has a therapeutic effect once uncoupled from the functionalprotein.
 7. The composition of claim 1 wherein the peptide mask is 8-15amino acids in length.
 8. The composition of claim 1 wherein the peptidemask has less than 50% amino acid sequence homology to its bindingpartner. 9.-10. (canceled)
 11. The composition of claim 1 wherein thefunctional protein is a full-length protein, a functional fragment of afull-length protein, a globular protein, a fibrous protein, or amultimeric protein.
 12. The composition of claim 1 wherein thefunctional protein is a ligand.
 13. The composition of claim 12 whereinthe ligand is an interferon protein.
 14. The composition of claim 13wherein the interferon protein is selected from the group consisting ofinterferon type I, interferon type II, and interferon type III.
 15. Thecomposition of claim 13 wherein interferon protein is selected from thegroup consisting of IFN-α, IFN-β, IFN-ω and IFN-γ.
 16. The compositionof claim 13 wherein interferon protein is IFN-α.
 17. The composition ofclaim 16 wherein the peptide mask contains a sequence selected fromthose presented in Table 3 or a sequence at least having 90% homologythereof.
 18. The composition of claim 16 wherein the peptide maskcontains the consensus sequence TDVDYYREWXXXXXXXX (SEQ ID NO: 1). 19.The composition of claim 16 wherein IFN-α protein is selected from thegroup consisting of 2a, 2b, and con1.
 20. The composition of claim 13wherein the binding partner is a receptor for the interferon protein.21. The composition of claim 20 wherein the receptor for the interferonprotein is selected from the group consisting of IFNAR, IFNAR1, IFNAR2,IFNGR, and IFNLR1.
 22. The composition of claim 1 wherein the functionalprotein is a soluble membrane protein or a functional fragment thereof.23. The composition of claim 1 wherein the functional protein is asoluble receptor or fragment thereof.
 24. The composition of claim 1wherein the functional protein is the extracellular domain of a receptorprotein or a fraction thereof.
 25. The composition of claim 23 whereinthe peptide mask inhibits the binding of the soluble receptor to itsligand.
 26. The composition of claim 25 wherein the peptide maskinhibits the receptor's ligand binding domain.
 27. The composition ofclaim 23 wherein the receptor is Notch.
 28. The composition of claim 27wherein the Notch receptor is selected from the group consisting Notch1,Notch2, Notch3 and Notch4.
 29. The composition of claim 25 wherein theligand is selected from the group consisting DLL1, DLL3, DLL4, Jagged1,and Jagged2.
 30. The composition of claim 27 wherein the peptide maskcontains a sequence selected from those presented in Table 14 or asequence having at least 90% homology thereof.
 31. The composition ofclaim 2 wherein the cleavable linker is a substrate for an enzymeselected from the substrates in Table
 2. 32. The composition of claim 2wherein the cleavable linker is a substrate for an enzyme selected fromthe group consisting of matriptase, plasmin, MMP-9, uPA, HCV-NS3/4, PSA,and legumain.
 33. The composition of claim 32 wherein the cleavablelinker is a substrate for matriptase or HCV-NS3/4.
 34. The compositionof claim 32 wherein the consensus sequence for a matriptase substratecomprises XXQAR(A/V)X or AGPR (SEQ ID NO: 2).
 35. The composition ofclaim 32 wherein the consensus sequence for a HCV-NS3/4 substratecomprises DEXXXC(A/S) or DLXXXT(A/S).
 36. The composition of claim 32wherein a sequence for an MMP-9 substrate comprises VHMPLGFLGP (SEQ IDNO: 3).
 37. The composition of claim 32 wherein a sequence for a plasminsubstrate comprises QGPMFKSLWD (SEQ ID NO: 4).
 38. The composition ofclaim 1 further comprising an Fc region of an immunoglobulin.
 39. Thecomposition of claim 1 wherein the coupling of the peptide mask to thefunctional protein is non-covalent.
 40. The composition of claim 1wherein the peptide mask inhibits binding of the functional protein toits binding partner allosterically.
 41. The composition of claim 1wherein the peptide mask inhibits binding of the functional protein toits binding partner sterically.
 42. The composition of claim 1 whereinthe binding affinity of the peptide mask to the functional protein isless than the binding affinity of the binding partner to the functionalprotein.
 43. The composition of claim 1 wherein the dissociationconstant (K_(d)) of the peptide mask towards the functional protein isat least 100 times greater than the K_(d) of the functional proteintowards its binding partner.
 44. The composition of claim 43 wherein theK_(d) of the peptide mask towards the functional protein is lower thanabout 5 nM.
 45. The composition of claim 3 wherein when the compositionis not in the presence of an enzyme capable of cleaving the cleavablelinker, the peptide mask inhibits the binding of the functional proteinto its binding partner by at least 90% when compared to when thecomposition is in the presence of the enzyme capable of cleaving thecleavable linker and the peptide mask does not inhibit the binding ofthe functional protein to its binding partner.
 46. The composition ofclaim 3 wherein the cleavable linker is capable of being specificallycleaved by an enzyme at a rate of at least 5×10⁴ M⁻¹S.
 47. (canceled)48. A method of treating a disease or disorder, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising: a. a pharmaceuticallyacceptable excipient; and b. a functional protein coupled to a peptidemask and a cleavable linker, wherein: i. the functional protein is notan antibody or an antibody fragment; ii. the peptide mask inhibitsbinding of the functional protein to its binding partner and does nothave an amino acid sequence of the binding partner, and iii. thecleavable linker is capable of being cleaved, such that in an uncleavedstate, the peptide mask inhibits binding of the functional protein toits binding partner and in a cleaved state, the peptide mask does notinhibit binding of the functional protein to its binding partner. 49.The method of claim 48 wherein the disease or disorder is cancer. 50.The method of claim 48 wherein the disease or disorder is Hepatitis Cinfection or hepatocellular cancer. 51.-70. (canceled)
 71. A modifiedprotein comprising a substrate capable of cleavage wherein the proteinis IFN-α and the substrate is capable of cleavage by matriptase orHCV-NS3/4, or wherein the protein is a soluble Notch receptor and thesubstrate is capable of cleavage by plasmin, legumain, PSA, or uPA.72.-77. (canceled)
 78. A protein therapeutic for the treatment ofHepatitis C having an improved bioavailability comprising a functionalprotein coupled to a peptide mask and a cleavable linker, wherein theaffinity of binding of the protein therapeutic to its target is higherin liver tissue when compared to the binding of the protein therapeuticto its target in a non-liver tissue, wherein target is present in bothtissues.
 79. The protein therapeutic of claim 78 wherein the cleavablelinker comprises substrate specific for a matriptase or HCV NS3/4enzyme.